Sczyrba A et al. (2005), XenDB: full length cDNA prediction and cross sp...

XB-ART-1369

BMC Genomics 2005 Apr 01;6:123. doi: 10.1186/1471-2164-6-123.

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XenDB: full length cDNA prediction and cross species mapping in Xenopus laevis.

Sczyrba A , Beckstette M , Brivanlou AH , Giegerich R , Altmann CR .

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Research using the model system Xenopus laevis has provided critical insights into the mechanisms of early vertebrate development and cell biology. Large scale sequencing efforts have provided an increasingly important resource for researchers. To provide full advantage of the available sequence, we have analyzed 350,468 Xenopus laevis Expressed Sequence Tags (ESTs) both to identify full length protein encoding sequences and to develop a unique database system to support comparative approaches between X. laevis and other model systems. Using a suffix array based clustering approach, we have identified 25,971 clusters and 40,877 singleton sequences. Generation of a consensus sequence for each cluster resulted in 31,353 tentative contig and 4,801 singleton sequences. Using both BLASTX and FASTY comparison to five model organisms and the NR protein database, more than 15,000 sequences are predicted to encode full length proteins and these have been matched to publicly available IMAGE clones when available. Each sequence has been compared to the KOG database and approximately 67% of the sequences have been assigned a putative functional category. Based on sequence homology to mouse and human, putative GO annotations have been determined. The results of the analysis have been stored in a publicly available database XenDB http://bibiserv.techfak.uni-bielefeld.de/xendb/. A unique capability of the database is the ability to batch upload cross species queries to identify potential Xenopus homologues and their associated full length clones. Examples are provided including mapping of microarray results and application of 'in silico' analysis. The ability to quickly translate the results of various species into 'Xenopus-centric' information should greatly enhance comparative embryological approaches.

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Species referenced: Xenopus laevis
Genes referenced: gnao1 zbtb7b

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	Figure 3. Identification of chimeric TCs: Matches of at least 100 bp in length were mapped back to the TC sequences to identify the regions that are covered by a match (yellow boxes). If two matches overlap, the region will be extended accordingly. If after the mapping two clearly separated regions remain as shown here, the TC is flagged as potential chimera.
	Figure 1. Full length clone selection (top) and TC categories (bottom). ESTs derived from different clones were clustered and assembled. The CAP3 contig was compared to protein databases using BLASTX and FASTY and hits categorized in 4 categories. Class 1 hits had to match the whole protein sequence and start with an ATG in the TC and M in the protein and the hit had to end at a STOP codon. Class 2 hits had to match the whole protein sequence, start with an ATG in the TC and M in the protein. Class 3 had to match the full protein sequence (without further restrictions), class 4 had to cover the protein over almost its full length, allowing the match to start or end maximal 10 ten amino acids after/before the start or end of the protein. Predicted 5' TCs (P5P) had to have enough sequence to fill up the missing 5' end of the protein sequence. Clone selection: Clone A and B were discarded because of missing IMAGE id. Clone 54321 does not span 5' end of protein match. Clone 21345 was selected as most 5' clone fulfilling the requirements.
	Figure 2. Comparison of a BLASTX alignment with corresponding full length FASTY alignment, as generated by the Genlight system. Blue boxes in (a) indicate open reading frames, green boxes start and red boxes stop codons, respectively. The assembled TC sequence has a frameshift at position 1150 from frame 1 to 3, generating two distinct HSPs in the BLASTX alignment (b). FASTY clearly corrects this frameshift and generates a full length alignment (c).
	Figure 4. Two examples of TCs derived from clones predicted to have a full length insert (P5P). The start positions in the hit suggest that the unmatched amino-terminal protein sequence is not well conserved between X. laevis and the matched organisms, here rabbit (top) and human (bottom), but the open reading frames (blue boxes) indicate that the clones the sequences were derived from do actually contain a full length insert. (Screenshots of the results were generated by the Genlight system.)
	Figure 5. Cluster view of the XenDB Web interface. Best FASTY hits to NR protein database, five model organisms and Xenopus proteins are shown on top. Gene Ontologies (GO) are based on best human and mouse IPI hits, functional categories on hits to COG and KOG databases. Below, additional information for each EST in the cluster is shown, such as accession, UniGene and TGI id, clone, cell and tissue type. Clones predicted not to be full length are colored red. Links to CAP3 assembly and TC sequence are provided.

References [+] :

Aaronson, Toward the development of a gene index to the human genome: an assessment of the nature of high-throughput EST sequence data. 1996, Pubmed