Exploring nervous system transcriptomes duringembryogenesis and metamorphosis in Xenopus tropicalisusing EST analysis.Ana C Fierro, Raphae¨l Thuret, Laurent Coen, Muriel Perron, Barbara ADemeneix, Maurice Wegnez, Gabor Gyapay, Jean Weissenbach, PatrickWincker, Andre´ Mazabraud, et al.To cite this version:Ana C Fierro, Raphae¨l Thuret, Laurent Coen, Muriel Perron, Barbara A Demeneix, etal.. Exploring nervous system transcriptomes during embryogenesis and metamorphosis inXenopus tropicalis using EST analysis.. BMC Genomics, BioMed Central, 2007, 8, pp.118.<10.1186/1471-2164-8-118>. HAL Id: hal-00159690https://hal.archives-ouvertes.fr/hal-00159690Submitted on 4 Jul 2007HAL is a multi-disciplinary open accessarchive for the deposit and dissemination of sci-entific research documents, whether they are pub-lished or not. The documents may come fromteaching and research institutions in France orabroad, or from public or private research centers.L’archive ouverte pluridisciplinaire HAL, estdestine´e au de´poˆt et a` la diffusion de documentsscientifiques de niveau recherche, publie´s ou non,e´manant des e´tablissements d’enseignement et derecherche franc¸ais ou e´trangers, des laboratoirespublics ou prive´s.BioMed CentralPage 1 of 13(page number not for citation purposes)BMC GenomicsOpen AccessResearch articleExploring nervous system transcriptomes during embryogenesis and metamorphosis in Xenopus tropicalis using EST analysisAna C Fierro*1,2,5, Raphaël Thuret1,2, Laurent Coen3, Muriel Perron1,2, Barbara A Demeneix3, Maurice Wegnez1,2, Gabor Gyapay4, Jean Weissenbach4, Patrick Wincker4, André Mazabraud1,2 and Nicolas Pollet*1,2,5Address: 1CNRS UMR 8080, F-91405 Orsay, France, 2Univ Paris Sud, F-91405 Orsay, France, 3CNRS UMR 5166, Evolution des Régulations Endocriniennes, USM 501, Département Régulations, Développement et Diversité Moléculaire, Muséum National d'Histoire Naturelle, 7 rue Cuvier, 75231 Paris Cedex 5, France, 4Genoscope and CNRS UMR 8030, 2 rue Gaston Crémieux CP5706, 91057 Evry, France and 5Programme d'Épigénomique, Univ Evry, Tour Évry 2, 10è étage, 523 Terrasses de l'Agora, 91034 Evry cedex, FranceEmail: Ana C Fierro* - carolina.fierro@gmail.com; Raphaël Thuret - raphael.thuret@u-psud.fr; Laurent Coen - coen@mnhn.fr; Muriel Perron - muriel.perron@u-psud.fr; Barbara A Demeneix - demeneix@mnhn.fr; Maurice Wegnez - maurice.wegnez@u-psud.fr; Gabor Gyapay - gabor@genoscope.cns.fr; Jean Weissenbach - jsbach@genoscope.cns.fr; Patrick Wincker - pwincker@genoscope.cns.fr; André Mazabraud - andre.mazabraud@u-psud.fr; Nicolas Pollet* - Nicolas.Pollet@u-psud.fr* Corresponding authors AbstractBackground: The western African clawed frog Xenopus tropicalis is an anuran amphibian species now usedas model in vertebrate comparative genomics. It provides the same advantages as Xenopus laevis but isdiploid and has a smaller genome of 1.7 Gbp. Therefore X. tropicalis is more amenable to systematictranscriptome surveys. We initiated a large-scale partial cDNA sequencing project to provide a functionalgenomics resource on genes expressed in the nervous system during early embryogenesis andmetamorphosis in X. tropicalis.Results: A gene index was defined and analysed after the collection of over 48,785 high quality sequences.These partial cDNA sequences were obtained from an embryonic head and retina library (30,272sequences) and from a metamorphic brain and spinal cord library (27,602 sequences). These ESTs areestimated to represent 9,693 transcripts derived from an estimated 6,000 genes. Comparison of thesecDNA sequences with protein databases indicates that 46% contain their start codon. Further annotationincluded Gene Ontology functional classification, InterPro domain analysis, alternative splicing and non-coding RNA identification. Gene expression profiles were derived from EST counts and used to definetranscripts specific to metamorphic stages of development. Moreover, these ESTs allowed identificationof a set of 225 polymorphic microsatellites that can be used as genetic markers.Conclusion: These cDNA sequences permit in silico cloning of numerous genes and will facilitate studiesaimed at deciphering the roles of cognate genes expressed in the nervous system during neuraldevelopment and metamorphosis. The genomic resources developed to study X. tropicalis biology willaccelerate exploration of amphibian physiology and genetics. In particular, the model will facilitate analysisof key questions related to anuran embryogenesis and metamorphosis and its associated regulatoryprocesses.Published: 16 May 2007BMC Genomics 2007, 8:118 doi:10.1186/1471-2164-8-118Received: 17 November 2006Accepted: 16 May 2007This article is available from: http://www.biomedcentral.com/1471-2164/8/118© 2007 Fierro et al; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.BMC Genomics 2007, 8:118 http://www.biomedcentral.com/1471-2164/8/118Page 2 of 13(page number not for citation purposes)BackgroundXenopus tropicalis is now an anuran amphibian referencegenome for vertebrate comparative genomics. It presentsthe same advantages as Xenopus laevis but has a smallergenome of 1.7 Gbp and a shorter generation time [1].Moreover, while X. laevis is an allotetraploid derived froman allopolyploidization event, X. tropicalis is diploid [2,3].Even though phylogenetic studies indicate that 30 to 50MY evolution separate the two species [3,4], it has beenshown that most methods and resources developed for X.laevis can be readily applied to X. tropicalis [5]. Thus, thegenome of X. tropicalis was selected to explore amphibiangenome characteristics by whole-genome shotgunsequencing [6].Working on X. laevis constitutes a challenge when dealingwith large-scale transcriptomics, such as microarraysexperiments or systematic cDNA sequencing. This isbecause some X. laevis genes are present as diploids, whileothers form pairs of paralogs (also called "pseudoalleles")that have been conserved with various degrees of diver-gence, generally less than 10% [7]. On a genomic scale,recent data has led to the estimation of 12% as the mini-mal fraction of paralogous gene pairs kept after allotetra-ploidization [8]. However, this estimate is based on theapplication of strict and conservative criteria: less than98% nucleotidic similarity and 93% mean similaritybetween paralogs. Therefore, it is likely that more than12% of paralogs are indeed active genes in X. laevis. More-over, such pairs of genes may have distinct expression pat-terns [7]. An estimated 14% of paralogs show distinctexpression profiles based on EST counts [8]. Given thesecomplications, it follows that the X. tropicalis genome ismore amenable to systematic transcriptome surveys thanthat of X. laevisTranscriptome analysis relies heavily on cDNA analysis.Collections of cDNA sequences have multiple uses for themolecular geneticist. They can be used to establish tran-script catalogues [9-11] and to provide experimental evi-dence when building gene models from genomicsequence, particularly for 5' and 3' untranslated sequences[12]. Further, they can be used to provide global views ongenome expression in a given cell type by the estimationof the abundance of the different mRNA species (throughsignatures as in [13]) and therefore can help decipherphysiological roles played by a given gene product.Finally, partial cDNA sequences (ESTs) are used to iden-tify full-length clones containing the entire open-readingframe for each transcript [14].We initiated an EST program so as to provide a functionalgenomics resource for X. tropicalis containing sequencesfrom the highest possible number of genes expressed inthe nervous system. We report the construction of such agene index and its assessment after the collection of48.785 partial cDNA sequences. These ESTs are estimatedto represent 6,000 genes that were annotated throughsequence similarity searches, protein domain searchesand Gene Ontology functional classification. Geneexpression profiles were derived from EST counts andused to evidence transcripts differentially expressed atmetamorphic stages of development. A set of polymor-phic intragenic microsatellite markers was deduced fromthe analysis of ESTs derived from distinct strains of X. trop-icalis. We expect that this resource will be valuable for fur-ther molecular genetics experiments.Results and discussionConstruction of cDNA libraries and normalizationTwo X. tropicalis cDNA libraries were constructed for thisproject. The first, designated xthr, was derived from dis-sected retinas and heads of young tadpoles (Nieuwkoopand Faber st. 25–35). About 500 retinas were dissectedfrom stage 32 X. tropicalis embryos, a stage where differen-tiating retinal neurons are getting organized into layers.Because these retinas yielded only few polyA+ RNA, thelibrary was enriched by the addition of mRNA from headsof embryos of the same developmental stage. The secondlibrary, designated xtbs, was made from central nervoussystems of metamorphosing tadpoles. Brains and spinalcords were dissected from tadpoles between stage 58 and64, the period covering the whole of Xenopus metamor-phosis. To build the library, and with the aim of respect-ing the relative proportion of nervous tissue obtained atthe different stages, samples for six animals were pooledfor each stage between 58–61 and three animals for eachstage between 62–64. All these tissues were combined andthe mRNA extracted for preparation of the xtbs library.The SMART technology (Clontech) was used to enrich therepresentation of full-length cDNA clones (defined hereas a copy of the transcript sequences between the 5' capand a polyA tail).To increase the information derived from EST projects, itis necessary to sample complex or normalised cDNAlibraries with few overrepresented cDNA clones (observedindividually with a frequency greater than 1%). To evalu-ate our libraries quality, samples of 1,989 cDNAs fromxthr and 1,694 cDNA from xtbs were partially sequenced(see Methods) to obtain 4,120 ESTs. Next, a normaliza-tion step was performed to increase the diversity ofsequence tags. We used a set of 53 oligonucleotides (35mers) corresponding to highly represented clones (≥ 1%,see Methods) in hybridizations on high-density colonyfilters (See additional file 1). A total of 22,561 clones werescored as positives (20% in both libraries) with an esti-mated false positive level of 0 and 3%, and an estimatedfalse negative level of 38 and 10% for xthr and xtbs librar-ies, respectively. The negatively scored clones were re-BMC Genomics 2007, 8:118 http://www.biomedcentral.com/1471-2164/8/118Page 3 of 13(page number not for citation purposes)arrayed to further the project using both 5' and 3' sequenc-ing. The further sequencing of cDNA clones provided48,785 high-quality sequences derived from 27,806clones after trimming 57,874 reads (including the 4,120ESTs of the pre-normalisation step, Table 1, see Methods).Both 5' and 3' end sequences were read for 75% of thecDNA clones, therefore reducing the difficulties associatedwith EST clustering. Moreover, this strategy helps to deter-mine the choice of a given cDNA clone for further experi-ments, whether it be full-length cDNA sequencing,overexpression studies or complementary RNA in vitrosynthesis.To determine if the normalization process was successful,the number of sequences containing each oligonucleotideprobe was counted before and after normalization (Fig.1). Before normalization, the 53 clusters from which theprobes were derived accounted for 18% of the 4,120 ESTs.This fraction dropped to 1% after normalization, confirm-ing the efficacy of the method. Of the 48 clusters corre-sponding to nuclear genes, 18 (37%) have 20 or morecorresponding ESTs and 17 (35%) have 40 or more ESTsafter normalization. We conclude that the abundance ofESTs after normalisation was sufficient in the majority ofcases. Even though this strategy requires re-arraying, thereis no bias due to insert length compared to normalizationby re-association [15] and therefore constitutes a usefulalternative.EST assemblyWe analyzed these sequences with PHRAP [16] to buildcontigs out of the overlapping and redundant sequences(Table 1). A total of 31,767 sequences were assembledinto 8,756 contigs. These were further grouped by virtueof clone links into 6,547 unique groups (scaffolds). Tak-ing into account the 2,982 singletons issued from 2,304clones, a total of 9,693 transcripts sequences were identi-fied. We compared our results to the global clustering ofall X. tropicalis ESTs (including ours) by the UniGene pipe-line and the DFCI Gene Index. In UniGene, our set of ESTsbelong to 7,778 groups made of between 1 and 220clones. Similarly, The DFCI Xenopus tropicalis Gene Indexclustered these ESTs in 9,350 TCs and 1,160 singletons.The majority of clusters (66%) contained three or lessESTs. Only 11 contigs were composed of more than 100sequences (See Additional file 2) and the largest contigcontained 159 sequences. Most of the corresponding geneproducts (23/50) are ribosomal proteins, the other beingproteins involved in basic cellular processes (tubulin,elongation factor 1 alpha). Two noteworthy exceptionsare myelin basic protein (contig8746) and metal-lothionein (contig8708), for which transcripts are foundalmost exclusively in the nervous sytem.The sequence redundancy (number of ESTs/cluster) ofxthr and xtbs libraries was compared to other X. tropicaliscDNA libraries represented in dbEST (See Additional file3). A statistically significant difference at the 1% level ofsignificance indicates that the complexity is higher foradult-type cDNA libraries, whether or not a size fraction-ation was performed. Amongst cDNA libraries preparedfrom embryonic or larval stages of development, the com-plexity of the xtbs library ranks first, while the complexityof the xthr library is close to the mean value.Sequences were assembled into contigs of up to 3 kb insize (hsp90 transcript, Contig 8575, See Additional file 4),but the mean contig length of 745 bp indicated that mostof them cover only parts of the cognate transcriptsequence.To assess the fraction of clones likely to be full-length, weestimated the number of sequences in our dataset thatTable 1: Xenopus tropicalis EST project statisticsxthr xtbs xthr and xtbsNumber of sequences reads obtained 30272 27602 57874Number of clone sequences obtained 16548 14901 31449Number of valid sequences 26440 22345 48785Number of clones with valid sequences 15540 12266 27806Number of clones with 5' and 3' EST 9354 11486 20840Number of clones with 5' EST only 4831 0 4831Number of clones with 3' EST only 1355 780 2135Average trimmed EST length 522 546 534Number of contigs 4327 4002 8756Number of contigs groups 497 289 842Number of contigs grouped 1210 649 2209Number of unique contigs 3117 3353 6547Nulmber of clones in contigs 9616 9268 15642Number of singletons 7958 4262 17018Number of clones in singletons 5924 2998 12164Number of putative transcripts 9538 6640 19553Max. assembled sequence length 3028 3144 3028Average assembled sequence length 732 782 745Max. assembled sequence size 147 144 159Average assembled sequence size 6 5 5Number of contigs containing1 509 97 11522 1779 2155 37303 452 362 9054–5 587 545 11596–10 505 476 95211–20 283 207 47921–30 91 90 17431–50 80 41 11850–100 31 26 76>100 10 3 11BMC Genomics 2007, 8:118 http://www.biomedcentral.com/1471-2164/8/118Page 4 of 13(page number not for citation purposes)extends over the 5' or 3' end of complete cDNA sequences(Figure 2; 1,945 entries from the X. tropicalis Xenopus GeneCollection [8], Xt-XGC and 2,963 entries from the SangerInstitute [17]). Using conservative criteria, at least one5'EST was found to provide additional 5' upstream or 3'downstream sequence for 854 complete cDNAs (17.4% ofthe set). Using the same criteria but only on contigsequences, further sequence information was obtained on355 complete cDNAs (7.2% of the set). Of these full-length cDNAs, 82 are completely matched by 122 contigs,and the latter are all longer. These results provide an indi-cation of the added-value of this sequence resource in theframework of the delineation of gene structure, especiallywith respect to the determination of the transcription ini-tiation site.Another way to assess the fraction of clones likely to befull-length has been described by Gilchrist et al. [17].Using this method on all X. tropicalis cDNA sequences(version Xt6 [18]) xthr and xtbs libraries we found to con-tain respectively 42% and 37% of full-length clones (MJGilchrist, personal communication). The mean fraction offull-length clones across all libraries is 18%. Therefore, weconclude that our normalization procedure did notimpair the proportion of full-length clones compared tonon-normalized libraries.Sequence annotationsIn order to further analyse our dataset, we compared ourcontigs to ENSEMBL predicted transcripts. Altogether,4,437 contigs (52%) and 1,423 singletons (48%)matched 4,083 transcripts from 3,703 ENSEMBL pre-dicted genes (15%). The extent of the underclustering ofour ESTs was estimated from these numbers and used tocalculate that our whole EST set represents about 6,000genes. We conclude that our cDNA sequence collectionsignificantly improved annotation of the X. tropicalisgenome sequence. Similarly, we compared our dataset toAssessment of normalization effectivenessFigure 1Assessment of normalization effectiveness. Histogram showing the percentage of sequence matching each oligonucle-otide used in the procedure of normalization by hybridization. Bars represent percentages of positive clones calculated before (grey bars) and after normalization (black bars). Data before normalization were obtained after partial sequencing of 1,989 cDNAs from xthr and 1,694 cDNAs from xtbs. Note the relatively high abundance of cell-type specific transcript such as gamma crystallin (crystallinG1) or neurogranin (underlined on the figure).1.55%0.92%0.87%BMC Genomics 2007, 8:118 http://www.biomedcentral.com/1471-2164/8/118Page 5 of 13(page number not for citation purposes)2,402 X. tropicalis RefSeq mRNA sequences. We found that2,230 contigs (26%) and 484 (16%) singletons matched1,342 RefSeq entries (56%). These figures suggest that fur-ther extensive sequencing of putative full-length cDNAclones from our collection will be of great use in order tocover the entire Xenopus gene set.We next estimated the proportion of our cDNA sequencesrepresenting mRNA molecules produced by a splicingevent and hence most likely to correspond to physiologi-cal products. We used "exonerate" to compute alignmentsbetween cDNA and genomic sequences. We retained onlythe alignments satisfying the thresholds of 95% identityand 90% coverage. Evidence of splicing was found for5,025 contigs (65%) out of 7,718 contigs aligned to thegenome. From the 2,693 contigs left, only 274 are signifi-cantly similar to a protein sequence and it is likely that theothers represent 3' untranslated regions, often encoded bya single exon in vertebrates.Next, two complimentary methods were used to find evi-dence for alternative splicing. Using genomic sequences(see Methods) we predicted 111 cases of alternative splic-ing, including conserved ones such as Clathrin light chain(contig7735 and contig8467, See Additional file 5). Usingalignments on protein and genomic sequences we found58 cases (such as elrD represented by contig7817, SeeAdditional file 6).Our set of transcript sequences was annotated using simi-larity searches in nucleotidic and protein databases andmotif searches (Table 2). Of the 8,756 contigs, 62% havemore than 70% nucleotidic similarity to previouslydescribed X. laevis regular entries, and may be consideredas "known" Xenopus genes. Of these sequences, 4,426 hadsignificant similarity to 2,803 protein sequences in Swiss-Prot database, and 5,506 to 3,571 cluster of the Uniref90database. We identified 212 sequences corresponding tothe Xenopus orthologs of human disease genes (See Addi-tional file 7). Further molecular studies on these genes inXenopus will be useful for understanding the physiopa-thology of these diseases.Putative coding regions were identified using frame-search, and corresponding protein sequences were anno-tated using InterProScan, allowing for an automatic GeneOntology Annotation (Table 3).Several known genes specifically expressed in the eye wereidentified, including different crystallins (beta, gammaand mu isoforms), vsx1 (visual system homeobox 1), pax6(paired-box protein 6), rdgb (retinal degeneration Bhomolog), rgr (RPE-retinal G protein-coupled receptor).Well-characterised central nervous system specific geneswere identified as well, notably elrC, mbp (myelin basicprotein), plp (myelin proteolipid protein 1). The corre-sponding cDNAs will provide useful differentiation mark-ers for X. tropicalis.A significant number of the contigs (37%) had no signifi-cant similarities to previously described genes, and mayrepresent transcribed pseudogenes, non-coding RNAsequences and undescribed genes. Indeed, comparing oursequences to non-coding RNA sequences (microRNAfrom RFAM, or ncRNA from the H-INV datasets) we found2 microRNA precursors (contig7127 and 7850 encodingmir-9-1 and mir-124a respectively) as well as E3(Contig2965) and 5SN4 (Contig5668) snoRNAs.Contig7127 (452 nt) is derived from the assembly of 6ESTs derived from 3 distinct cDNA clones of the xtbslibrary. The alignment of contig7127 sequence on X. trop-icalis genome sequence reveals 100% identity and indi-cates that one splicing event is required. Thus, contig7127represents a bona fide neural transcript of the mir-9-1gene. Contig7850 (800 nt) is derived from the assemblyof 10 ESTs derived from 6 cDNA clones (one from xtbsand 5 from xthr libraries). Four of these clones are identi-cal and characterized by a 409 bp cDNA, while two arelonger and have their 3' ends ESTs as singletons but map-ping to the same scaffold region.Added-value of xthr and xtbs 5'ESTsFigure 2Added-value of xthr and xtbs 5'ESTs. 5' cDNA sequences were compared to 4,908 complete X. tropicalis cDNA sequences from XGC and Sanger Institute. When an EST matched unambiguously (>95% id over more than 50 nt on the same orientation) one of these cDNAs, the position of its first residue (X axis) was plotted as a function of the cDNA size (Y axis). Each dot represents the result of an alignment. A position of 0 on the x axis indicates identical 5' ends between the EST and cDNA. Negative values indicate that the EST extends further 5', positive values superior to the cDNA length indicate that the EST extends further 3', and positive values inferior to the cDNA size indicate the 5' EST position relative to the cDNA.BMC Genomics 2007, 8:118 http://www.biomedcentral.com/1471-2164/8/118Page 6 of 13(page number not for citation purposes)Expression profilesOther collections of X. tropicalis ESTs are ongoing[8,17,19] using a variety of cDNA libraries made fromadult tissues or embryos at different stages of develop-ment. Hence, we undertook an in silico analysis of geneexpression profiles estimated from EST counts [20].In a first analysis, we searched transcripts identified byESTs derived predominantly from our cDNA libraries. Weidentified 99 and 238 cDNAs found prominently in theheads and retinas of tailbuds or brain and spinal cord oftadpole, respectively (See Additional file 8 and Additionalfile 9) and 25 clones found predominantly in both struc-tures. These clones are likely to represent genes differen-tially expressed in the retina or the central or peripheralnervous system during metamorphosis. The study of thesegenes in Xenopus could well improve our knowledge onCNS development and function in vertebrates.MetamorphosisIn a second analysis, we explored the metamorphosistranscriptomes using expression profiles derived from ESTcounts. It is known that amphibian metamorphosisbrings about unique regulations triggered by thyroid hor-mones during late vertebrate development, but relativelyfew genes are characterised as playing regulatory roles inthis process [21]. We extracted 4,187 UniGene clusterscontaining at least one EST from the xtbs cDNA library.Similarly, we fetched data from 592 UniGene clusters con-taining at least one and up to 132 ESTs from anotherlibrary derived from a metamorphic stage of develop-ment. Combining both sets gives 4,779 UniGene clusters(13% of all clusters). To generate a useful expressionmatrix an initial filtration step was performed wherebyclusters composed of less than 10 ESTs were removedleading to a set of 3,422 UniGene clusters. We used the GTtest [22] to rank profiles in three categories: strong (64clusters with GT > 0.66), medium (803 clusters with 0.33< GT < 0.66) or weak (2555 cluster with GT < 0.33) differ-ential expression. Because UniGene is prone to overclus-tering we focused our analysis on the 64 clusterscorresponding to genes with a strong differential expres-sion and analysed their expression profiles using hierar-chical clustering (Figure 3). Twelve characteristicexpression profiles are observed, corresponding to peaksof expression that are tissue (brain, intestine, kidney,heart, lung, skeletal muscle, skin) or stage-specific (egg,tailbud, tadpole, metamorphosis). The correspondinggenes are potential differentiation markers that can beuseful in developmental studies and can easily be checkedby in situ hybridization on embryos. Only one transcripttagged by ESTs derived solely from a metamorphic stagewas identified. This transcript codes for preprocaeruleintype-4; it is characterized by 40 ESTs derived from 24cDNA clones issued from 6 libraries made from stage 62and 64 tadpoles, i.e. representing late metamorphosisstages. Caerulein is a peptide found predominantly inskin secretions. It belongs to the gastrin/cholecystokininfamily of neuropeptide, and may play a role as an antimi-crobial molecule [23]. This finding is discussed later.Since there are currently ten times more ESTs in cDNAlibraries derived from metamorphic stages of develop-ment in X. laevis than in X. tropicalis, we did a similar sur-vey of the expression profiles of transcripts in X. laevis.We extracted 6,297 UniGene clusters (24% of all clusters)containing at least one and up to 710 ESTs from at leastone cDNA library prepared from metamorphic tadpoles.This corresponds to 24,262 ESTs made from four cDNAlibraries: limb, tail, intestine and tadpole (NF stage 62).The level of expression of each transcript was estimated bycounting ESTs providing a corresponding UniGene clus-ter. The 26 clusters containing the highest number ofESTs, and hence corresponding to the most highlyexpressed genes during metamorphosis, are listed in table4. We expected to find either ubiquitously or differentiallyexpressed categories among these highly abundant tran-scripts at metamorphosis stages. Indeed, 16 of these 25UniGene clusters are found as characterized by a restrictedexpression in the tail, limb, or heart (table 4). Interest-ingly, it is known that 11 are expressed in the muscle cellsthat compose most of the tail and limb. One remarkablecase is a gene coding for a protein involved in freeze-toler-Table 2: Results of sequence comparisonsBLASTX BLASTNSwissProt Uniref 90 X. laevis reg. entriesX. laevis UniGeneX. tropicalis UniGeneX. tropicalis genome X. tropicalis cDNAND 2034All hits 4426 51% 5506 63% 5447 62% 7175 82% 7865 90% 8703 99% 3877 44% All hits>= 90% 1594 18% 2753 31% 744 8% 864 10% 7199 82% 8371 96% 3660 42% >95%70 – 90% 1705 19% 1917 22% 2963 34% 3843 44% 454 5% 302 3% 217 2% 90 – 95%< 70% 1127 13% 836 10% 1740 20% 2468 28% 212 2% 30 0% 0 0% < 90%No similarity 4330 49% 3250 37% 3309 38% 1581 18% 891 10% 53 1% 4879 56% No similarityBMC Genomics 2007, 8:118 http://www.biomedcentral.com/1471-2164/8/118Page 7 of 13(page number not for citation purposes)ance found predominantly in metamorphic limbs, butexpressed in a variety of other tissues both embryonic(starting at gastrula stage) and adult (nearly all adult tis-sues sampled with the exception of ovary, testis and lung).This can be an artefact due to the handling of tissues at thetime of RNA extraction. Alternatively, this may reflect theinduction by stress-related hormones (glucocorticoids)during metamorphosis.We then carried out an in silico reconstruction of the tran-scriptional profile of X. laevis metamorphic genes usingthe IDEG suite of statistical tests. We removed clusterscomposed of less than 10 ESTs, leading to a set of 3,599UniGene clusters. The GT test ranked profiles in three cat-egories: strong (167 clusters with GT > 0.66) medium(1,300 clusters with 0.33 < GT < 0.66) or weak (2,132cluster with GT < 0.33) differential expression.From the 167 clusters corresponding to genes with astrong differential expression, only 30 are composed of atleast 2 ESTs derived solely from metamorphic or adult tis-sues, four of which bear no similarity to known proteins.We analysed the expression profiles of these metamorphicgenes using unsupervised hierarchical clustering. Theresulting clusters could be interpreted along the predomi-nant expression domains (See Additional file 10). Threeclusters (metamorphic, tadpole and limb) correspond tolarval stages of development that are made of eight, threeand four genes, respectively (Fig. 4). These genes arepromising candidates, potentially playing important rolesduring this late developmental event. Below, we describebriefly what is known about each of these genes.A larval beta chain of globin is among the metamorphiccluster together with an alpha chain, an indication of therelevance of our analysis. The comparison of Xl.56714EST sequences with known proteins shows that theyresemble cell surface receptors of the SLAM (SignallingLymphocytic Activation Molecule) family. The SLAMreceptors regulate immune cell activation. Indeed, it isknown that immune system remodelling is a major eventof metamorphosis [24]. The gene corresponding toXl.56714 is expressed in metamorphic tadpoles (includ-ing tail and intestine) as well as in the adult kidney. How-ever we could not detect significant similarities to anyknown gene sequences or proteins. Alpha-1 antichymot-rypsin (a plasma protease inhibitor) is highly expressedduring metamorphosis and found in adult liver. This cor-relates with the associated stress condition that occursduring tadpole transformations. The gene encodingalpha-2-HS-glycoprotein (also named fetuin) steadilyincreases in expression from tailbud stage up to metamor-phosis. This gene product is secreted in plasma and playsa physiological role during mammalian fetal develop-ment, especially in mineralization and growth. A knownXenopus gene encoding a small peptide named PYLa isfound exclusively in a cDNA library prepared from stage62 tadpoles. As for the preprocaerulein transcripts in X.tropicalis, the PYLa transcripts are abundant in metamor-phic stages, with ESTs found in limbs and whole tadpoles.Both caerulein and PYLa peptides may be secreted fromskin glands and exert antimicrobial activities. This findingcorroborates a previous report on caerulein expression[25]. Remarkably, skin glands are known to express acocktail of signalling peptides, including neuropeptidessuch as xenopsin, thyrotropin-releasing hormone andPGLa. Whether these peptides play specific roles in thecontext of metamorphosis is unknown. The clusterXl.24674 corresponds to a gene resembling uromodulin.Table 3: GO Molecular function classification.Gene ontology term NAll molecular function terms 1775->antioxidant activity 14->binding 623-->calcium ion binding 89-->carbohydrate binding 7-->lipid binding 10-->nucleic acid binding 350--->DNA binding 135---->chromatin binding 0---->transcription factor activity 31--->RNA binding 40--->translation factor activity, nucleic acid binding 58-->nucleotide binding 51-->oxygen binding 0-->protein binding 52--->cytoskeletal protein binding 25---->actin binding 23-->receptor binding 28->catalytic activity 455-->hydrolase activity 110--->nuclease activity 9--->peptidase activity 66--->phosphoprotein phosphatase activity 6-->kinase activity 56--->protein kinase activity 33-->transferase activity 112->chaperone regulator activity 0->enzyme regulator activity 37->motor activity 15->nutrient reservoir activity 0->signal transducer activity 48-->receptor activity 13-->receptor binding 28->structural molecule activity 398->transcription regulator activity 40->translation regulator activity 58->transporter activity 120-->electron transporter activity 39-->ion channel activity 11-->neurotransmitter transporter activity 3->triplet codon-amino acid adaptor activity 0BMC Genomics 2007, 8:118 http://www.biomedcentral.com/1471-2164/8/118Page 8 of 13(page number not for citation purposes)Digital expression profiles of X. tropicalis transcripts differentially expressed at metamorphosisFi ure 3Digital expression profiles of X. tropicalis transcripts differentially expressed at metamorphosis. Each line gives the expression profile of a given transcript represented by a UniGene cluster. The expression is deduced from counting the occurence of ESTs derived from a given cDNA library. The level of expression is colour coded in blue shades, dark blue means evidence for high levels of transcripts and white means no evidence for expression. On the left, clusters of expression profiles are delineated by a vertical bar labelled with the associated characteristic domain of expression. On the right, the cluster name and its annotation (i.e. the corresponding gene product description as deduced from sequence similarity analysis) are given. Each column corresponds to a category of tissue or stage of development: 8 adult tissues and 6 stages of development. Note that a given category may correspond to several cDNA libraries. Here, only clusters for which evidence of differential expres-sion were used to build the matrix of expression. This matrix was analysed by hierarchical clustering on the expression profile dimension using CLUSTER 3.0 as described in the methods section. adipose tissue brain digestive genitourinary head heart lung skeletal muscle skin egg gastrula neurula tailbud tadpole metamorphosis Str.20603 keratin 5 Str.40562 keratin 14 Str.49404 keratin 5 Str.41834 keratin 14 Str.51481 transcribed locus Str.41889 26 serine protease Str.41838 transcribed locus Str.51637 acyl-CoA dehydrogenase Str.29235 G protein pathway suppressor 2 Str.4962 slc7a7 Solute carrier family 7 Str.8295 elastase I Str.5512 ctrb2: chymotrypsinogen B2 Str.26623 lactotransferrin Str.26669 arginosuccinate synthetase (ass) Str.38826 apolipoprotein A-I precursor Str.7018 frzb: Frizzled-related protein (frzb) Str.8426 myl3: Myosin, light polypeptide 3 Str.24081 transcribed locus Str.2078 NADH dehydrogenase Str.49042 cytochrome-c oxidase Str.21355 progonadoliberin-2 precursor Str.22092 transcribed locus Str.49431 preprocaerulein type-4 Str.5804 kinesin light chain Str.17081 myosin heavy chain, fast skeletal muscle, embryo Str.8341 collagen, type V, alpha 3 Str.21728 transcribed locus Str.5815 DNA-binding protein A; Y box protein 3 Str.35909 myosin, heavy polypeptide 2, fast muscle Str.37351 ribosomal protein L3-like Str.27777 mogat2: Monoacylglycerol O-acyltransferase 2 Str.3439 eukaryotic translation initiation factor 2-alpha Str.21815 erythrocyte membrane protein band 4.9 (dematin) Str.28019 discs, large homolog 4 Str.14347 RAB40B, member RAS oncogene family Str.21084 C14orf37 Str.21253 transcribed locus Str.52283 Rho-related GTP-binding protein RhoI Str.21114 neurensin 1 Str.21299 phytanoyl-CoA 2-hydroxylase interacting protein Str.20386 myelin basic protein Str.14909 ADP-ribosylation factor 3 Str.28128 microtubule-associated protein 2 Str.21155 neuronal pentraxin II Str.21068 transcribed locus Str.21392 transmembrane protein 59 Str.28868 centaurin gamma 1 Str.21176 neurexin 1, isoform alpha Str.28022 fibrinogen C domain containing 1 Str.20925 protein tyrosine phosphatase, receptor type, N Str.21977 kinesin-like protein KIF1A Str.15361 arrestin beta 1, isoform B Str.38310 latrophilin 1 Str.20880 contactin associated protein 1 Str.21943 transcribed locus Str.21088 EPH receptor A8 Str.21930 transcribed locus Str.8446 EF-hand calcium binding protein 1 Str.38334 lysosomal associated protein transmembrane 4 beta Str.49508 myelin proteolipid protein Str.26607 C2orf21 Str.21007 glutamate receptor, ionotrophic, AMPA 4 Str.38154 calcium channel, voltage-dependent, alpha 2/delta 1 Str.5352 prickle-like 2 skinkidneyintestinehearttailbudtadpolemetamorphmusclelungbrainBMC Genomics 2007, 8:118http://www.biomedcentral.com/1471-2164/8/118Page 9 of 13(page number not for citation purposes)Table 4: Highly expressed metamorphic transcripts.UniGene ID Met ESTs Total ESTs cDNA source Note Restricted expression DescriptionXl.24656_a 710 1405 b;h;l;li;ot;sk;t;wb R ubiquitous tail actin alpha 1 skeletal muscleXl.1115__b 248 653 et;h;he;l;ot;sk;t;wb R tail, limb tail actin alpha 1 skeletal muscleXl.17432 560 4290 b;et;ey;fb;h;he;k;l;li;lu;ot;ov;s;sk;t;te;th;wb U ubiquitous - eukaryotic translation factor 1 alpha, somatic formXl.5860 397 1008 b;he;l;ot;s;sk;t;te;wb U ubiquitous - creatine kinase, muscleXl.11405 280 751 b;et;l;t;wb R tail, limb, tailbud tail Calcium ATPase at 60A, cardiac muscleXl.24815 238 510 b;l;t;th;wb R limb, tadpole limb myosin, heavy polypeptide 13, skeletal muscleXl.47042 227 468 b;et;ey;fb;he;k;l;li;s;sk;t;th;wb R limb metamorphosis collagen, type I, alpha 1Xl.25492 217 833 l;ot;sk;wb R limb, tadpole limb metamorphosis oncomodulinXl.7551 211 1333 b;et;ey;fb;h;he;k;l;li;lu;ot;ov;s;sk;t;te;th;wb U ubiquitous - eukaryotic translation elongation factor 2Xl.28832 205 474 et;h;h;l;t;wb R tail, limb tail metamorphosis actin alpha skeletal muscleXl.4138__a 198 1376 b;et;ey;fb;h;k;l;li;lu;ot;ov;s;sk;t;te;th;wb U ubiquitous - actin cytoplasmic 1Xl.29221_b 98 644 b;et;ey;fb;he;k;l;li;lu;ot;ov;s;sk;t;te;th;wb U thymus - actin cytoplasmic 1Xl.5146 168 360 b;et;ey;fb;he;k;l;li;s;t;th;wb R limb limb metamorphosis freeze tolerance-associated protein FR47Xl.8842 164 1033 b;et;ey;fb;h;he;k;l;li;lu;ot;ov;s;sk;t;te;th;wb N limb, thymus, spleen_PHA - ribosomal protein L3Xl.1032 158 243 b;l;ot;sk;t;wb R tadpole limb metamorphosis fast skeletal troponin C betaXl.1055 151 288 b;et;h;l;ot;sk;t;th;wb R limb, tailbud, tadpole limb metamorphosis myosin light chain 1, fast skeletal muscle isoformXl.24699 134 426 b;ey;he;l;lu;t;th;wb R limb, heart heart metamorphosis actin alpha cardiacXl.7875 120 975 b;et;ey;fb;h;he;k;l;lu;ot;ov;s;sk;t;te;th;wb D ubiquitous - solute carrier family 25 (mitochondrial carrier; adenine nucleotide translocator), member 5Xl.1728 120 884 b;et;ey;fb;h;he;k;l;li;lu;ot;ov;s;sk;t;te;th;wb N limb, tadpole, olfactory epith.- acidic ribosomal protein P0Xl.8672 116 756 b;et;ey;fb;h;he;k;l;li;lu;ot;ov;s;sk;t;te;th;wb N limb - ribosomal protein L4Xl.49126 115 272 b;ey;fb;l;lu;ot;s;sk;t;te;wb D ?ubiquitous limb metamorphosis procollagen, type I, alpha 2Xl.995 113 440 b;et;ey;fb;h;he;k;l;lu;ot;ov;s;sk;t;te;th;wb N limb - glyceraldehyde-3-phosphate dehydrogenaseXl.3463 109 554 b;et;ey;fb;h;he;k;l;li;lu;ot;ov;s;sk;t;te;th;wb R limb metamorphosis guanine nucleotide binding protein, beta 2, related sequence 1Xl.118 102 274 b;et;fb;h;k;l;lu;ot;s;sk;t;wb R limb metamorphosis tropomyosin 1 alpha chainXl.1464 100 537 fb;l;li;ot;s;sk;t;wb R tail, tadpole tail myosin, heavy polypeptide 4, skeletal muscleXl.395 94 479 li;lu;th;wb R liver liver serum albumin 74 kDaXl.938 94 221 b;ey;fb;l;ot;sk;t;te;wb R limb limb metamorphosis tropomyosin beta chain, skeletal muscleXl.23399 94 156 b;l;ot;sk;t;wb D ?ubiquitous tail metamorphosis aspartyl beta-hydroxylaseb: brain;et: embryonic tissue;ey: eye;fb: fat body;h: head;he: heart;k: kidney;l: limb;li: liver;lu: lung;ot: other;ov: ovary;s: spleen;sk:skin; t: tail;te: testis;th: thymus;wb: whole body. Allogenes are written in italics. U: ubiquitous; D: no differential; N: no restricted expression; R: restricted expression.BMC Genomics 2007, 8:118 http://www.biomedcentral.com/1471-2164/8/118Page 10 of 13(page number not for citation purposes)Corresponding transcripts are found in metamorphicintestine and whole tadpole. In mammals, uromodulin isexcreted in urine and plays a role in the cellular defenseresponse. A gene encoding a stomatin homolog is highlyexpressed in intestine during metamorphosis. Stomatin isa membrane protein regulating cation exchange andcytoskeletal attachment. Among the genes represented inthe metamorphic limb cluster are a keratin and two clus-ters (Xl.57017 and Xl.57064) annotated as lacking signif-icant similarities to known proteins. In the tadpolecluster, a carboxyl ester lipase is found expressed in tad-pole and in metamorphic intestine. Mucin 2 is anothergut protein highly expressed in tadpole, as well as carbox-ypeptidase.Taken together, these expression profiles, based on ESTcounts, reveals certain genes that are up-regulated duringmetamorphosis, possible targets of the thyroid hormonessignalling pathway.PolymorphismsThe cDNA sequences we produced are derived from theAdiopodoume strain of X. tropicalis, originating from theIvory Coast. The genomic and most other cDNA sequenc-ing efforts are made on the N strain from Nigeria or a dis-tinct IC strain from Ivory Coast. We therefore looked forpolymorphisms that could be used in genetic mappingexperiments or to discriminate with mutations obtainedfrom ENU mutagenesis. We identified 8 SNPs derivedfrom mitochondrial genes, three of which (snp1A, snp2A,snp6G) are specific to the Adiopodoume strain (See Addi-tional file 11). The presence of shared alleles for 5 SNPsindicates the close relationship with the N strain asalready reported by Evans et al. 2004. We searched fornovel polymorphism markers made of di, tri, tetra andpentanucleotide sequence repeats present in our EST col-lection. We found from two to ten alleles in 225 markersderived from 212 contigs/ESTs clusters. A subset of 107markers are potential highly informative since two ormore alleles are observed at high frequencies (See Addi-tional file 12). The dinucleotide repeat AT and TA are themost common, accounting for 137 markers. These intra-genic markers should be useful once placed on a geneticlinkage map.This dataset will provide an invaluable tool for exon defi-nition when the X. tropicalis genome sequence is finallydetermined. The results presented here are availablethrough a database on our web site [26]. Users can carryout BLAST and other searches based on GO classification,InterproScan results, and expression information. ThecDNA sequences have been deposited with Genbank/EMBL/DDBJ (accession numbers CN072222 –CN121006) and clones are available upon request.ConclusionLarge-scale cDNA sequencing has provided invaluableresources to decipher vertebrate genome structure andDigital expression profiles of X. laevis transcripts differentially expressed at metamorphosisFi ure 4Digital expression profiles of X. laevis transcripts differentially expressed at metamorphosis. Using the same rep-resentation as in Fig. 3 three clusters are depicted that are associated with differential expression at metamorphosis (top clus-ter), tadpole stage (middle) and in the forming limb (down). animal cap brain eye fat body genitourinary head heart limb liver lung lymphoreticular skin female germ line blastula gastrula gastrula/neurula cusp neurula tailbud tadpole metamorphosis Xl.56714 transcribed locus cDNA clone IMAGE:6871962 Xl.23966 Alpha-1-antichymotrypsin precursor Xl.24530 Xl Larval beta II globin Xl.1127 Xl Hemoglobin alpha chain Xl.24560 alpha-2-HS-glycoprotein Xl.144 Xl PYLa precursor Xl.24674 uromodulin Xl.26509 Xl stomatin Xl.51631 Keratin, type II cytoskeletal 1 Xl.57017 transcribed locus cDNA clone XL081o03 Xl.57064 transcribed locus cDNA clone IMAGE:8636 Xl.34936 Xl preprocaerulein type I Xl.24591 Xl carboxyl ester lipase Xl.24594 mucin 2 precursor, intestinal Xl.26001 carboxypeptidase B metamorphosistadpolelimbBMC Genomics 2007, 8:118 http://www.biomedcentral.com/1471-2164/8/118Page 11 of 13(page number not for citation purposes)function. Recent studies on cDNA sequencing with deepcoverage provide fundamental knowledge on the com-plexity of transcriptomes in mammals [27]. Here, we pro-vide information on the transcript sequence andexpression of an estimated 6,000 genes in X. tropicalis. Aweb resource [26] is available with associated annota-tions. The genetic resources stemming from the cDNAsequencing project described here can be used in diverseresearch projects, including vertebrate comparativegenomics, studies on evolution and development, cellbiology and developmental genetics. More specifically,retinogenesis and remodelling of the central nervoussytem during metamorphosis will benefit from this cDNAresource.We are currently undertaking full cDNA insert sequencingfor a set of non-redundant clones, as well as characterizingtheir expression using a whole-mount in situ hybridiza-tion screen [28,29]. The genomic resources developed tostudy X. tropicalis biology are crucial to explore amphibianphysiology and genetics, this model system providingexcellent characteristics for addressing key questionsrelated to anuran metamorphosis and its associated regu-latory processes.MethodsEmbryo and tissue dissectionEmbryos of Xenopus tropicalis Adiopodoumé strain wereobtained from parents issued of the Geneva collection[30].From each of the two libraries made, 58,368 clones werepicked, arrayed in microtiter plates and gridded on high-density nylon filters. A sample of 1,989 cDNA clones fromthe xthr library and 1,694 clones from xtbs were partiallysequenced to obtain 4,120 ESTs. This step provided aquality assessment of the two libraries, showing theabsence of clones of bacterial origin and few ribosomal(0.45% in xthr, 0% in xtbs) and mitochondrial contami-nants (1.2 and 3.6% in xthr and xtbs, respectively). Thisprocedure provided information on overrepresentedclones, which were then removed before further sequenc-ing of up to 30,000 clones. These ESTs were grouped into1,985 clusters.Normalization of cDNA librariesTwo pools of 25 and 28 oligonucleotides probes of 35 ntin length were labelled using Terminal Transferase (NewEngland Biolabs) and P33-dATP (Amersham). Labelledoligonucleotides were hybridised on two high-density fil-ters representing the xthr and xtbs libraries as in [31]. Pos-itive clones were identified using X-Digitize software onimages acquired using a phosphorimager.High-throughput sequencing, assemblingThe reactions were performed with a Big-Dye terminatorcycle sequencing kit and analyzed by ABI-3700 and ABI-3730. Sequences were base-called using PHRED, thentrimmed using LUCY and custom perl scripts. Sequencesless than 100 bp were discarded, as well as those identifiedto be derived from ribosomal and mitochondrial RNAs.PHRAP was used to assemble the sequences taking intoaccount quality scores, further clustering was obtained byscaffolding using mate-pairs informations. We retainedscaffolds only if two clone links were available (exceptingsingletons) and if the orientation of the reads was consist-ent.AnnotationRepetitive sequences were masked using CENSOR. Con-tigs and singletons were used as queries in BLASTX andBLASTN searches of Swissprot, Uniprot, Unigene (rel 70)and Xenopus tropicalis JGI assembly v4.1 databases onINFOBIOGEN server. The february 2005 release ofENSEMBL was used. Framefinder was used to identifycoding sequences, and protein domains were searchedusing INTERPROSCAN. The results of sequence assembly,scaffolding and annotation were loaded on a custom-made mySQL database. Web interface was developedusing PHP scripts.Assessment of the fraction of clones likely to be full-length5'ESTs or contigs were compared to full-length cDNAsusing BLASTN. Alignments longer than 50 nt and exhibit-ing more than 95% identities were selected. Overlapbetween query and subject sequences was scored onlywhen the alignment encompassed up to the last nucle-otide at the 5' or 3' end of the sequences.Alternative splicingAlignments between cDNA and genomic sequences(masked) were computed using exonerate. Evidence foralternative splicing was found when two contigs werealigned to the same genomic region with at least 95%mean overlap but with a different number of exons.Alignments between cDNA and UNIREF proteinsequences were computed using BLASTX. Alignmentscharacterized by a gap (at least 10 aa) introduced in thecontig sequence were retrieved. Alternative splicing wasconsidered present if the contig sequence including thegap could be aligned to the genomic sequence.Expression profilesEST counts were downloaded from UniGene (release 70for X. laevis and 32 for X. tropicalis). Relevant profiles wereextracted using custom perl scripts. GT test was run usingthe IDEG6 software [22]. Single hierarchical clusteringwas performed using Cluster 3.0 software [32]. We usedBMC Genomics 2007, 8:118 http://www.biomedcentral.com/1471-2164/8/118Page 12 of 13(page number not for citation purposes)absolute correlation similarity metrics followed by com-plete clustering on mean centered gene expression pro-files. Results were visualised using TreeView.PolymorphismsMitochondrial SNPs were identified on a collection ofmitochondrial ESTs downloaded from dbEST, JGI [33]and from our own set. These ESTs were assembled usingCAP3 and analyzed using visualization software [34].Microsatellites were identified using custom perl scripts.Authors' contributionsRT, LC and MP carried out laboratory and data analysis.AF wrote and ran the EST processing pipeline, includingEST assembly and annotation, and is responsible for theweb-available database. G.G., J.W. and P.W. managed andconducted the sequencing experiments. BD, MW and AMparticipated in the coordination of the study. NP partici-pated in the conception and design of the study, carriedout laboratory and data analysis and drafted the manu-script.Additional materialAdditional File 1Oligonucleotides used for normalization. The table provided lists the oligonucleotide identifier, corresponding gene, number of corresponding ESTs, oligonucleotide sequence and Tm.Click here for file[http://www.biomedcentral.com/content/supplementary/1471-2164-8-118-S1.xls]Additional File 2Top 50 of contigs according to the number of constituent ESTs. The table provided lists the Contig identifier, the number of constituent sequence reads, the contig length and the description of the best Swissprot hit identified by blastx.Click here for file[http://www.biomedcentral.com/content/supplementary/1471-2164-8-118-S2.xls]Additional File 3Analysis of X. tropicalis cDNA libraries complexity. The data provided represent the description and complexity of X. tropicalis cDNA libraries sampled by more than 20,000 ESTs.Click here for file[http://www.biomedcentral.com/content/supplementary/1471-2164-8-118-S3.xls]Additional File 4Top 50 of contigs according to their size. The table provided lists the Contig identifier, the contig length and the description of the best Swiss-prot hit identified by blastx.Click here for file[http://www.biomedcentral.com/content/supplementary/1471-2164-8-118-S4.xls]Additional File 5Alternative splicing detected by a different number of exons in contigs aligned in the same genomic region. The table provided lists the Contig identifier, the number of constituent exons, the description of the best pro-tein hit identified by blastx. Each line describes one alternative splicing case.Click here for file[http://www.biomedcentral.com/content/supplementary/1471-2164-8-118-S5.xls]Additional File 6Alternative splicing detected by a gap in the alignment against UniRef100. The table provided lists the Contig identifier, the identifier of the protein evidencing an alternative splicing event, and the identifier of the protein showing the highest similarity by blastx. Each line describes one alternative splicing case.Click here for file[http://www.biomedcentral.com/content/supplementary/1471-2164-8-118-S6.xls]Additional File 7Xenopus tropicalis genes related to human disease genes. The table provided lists the human disease trait, corresponding human gene name, chromosomal location, OMIM identifier and the corresponding Xenopus tropicalis Contig identifier.Click here for file[http://www.biomedcentral.com/content/supplementary/1471-2164-8-118-S7.xls]Additional File 8cDNA clones found specifically in library xthr. The table provided lists the Contig identifier and the description of the best protein hit identified by blastx.Click here for file[http://www.biomedcentral.com/content/supplementary/1471-2164-8-118-S8.xls]Additional File 9cDNA clones found specifically in library xtbs. The table provided lists the Contig identifier and the description of the best protein hit identified by blastx.Click here for file[http://www.biomedcentral.com/content/supplementary/1471-2164-8-118-S9.xls]Additional File 10Digital expression profiles of X. laevis transcripts. Using the same for-malism as in Fig. 3, all X. laevis Unigene clusters associated with differ-ential expression at metamorphosis are depicted.Click here for file[http://www.biomedcentral.com/content/supplementary/1471-2164-8-118-S10.eps]Additional File 11Mitochondrial SNPs. The table lists the occurence of given alleles of mito-chondrial SNPs in the adiopodoume and N strains, in association with the corresponding mitochondrial gene.Click here for file[http://www.biomedcentral.com/content/supplementary/1471-2164-8-118-S11.xls]BMC Genomics 2007, 8:118 http://www.biomedcentral.com/1471-2164/8/118Page 13 of 13(page number not for citation purposes)AcknowledgementsWe thank L. Du Pasquier for the gift of X. tropicalis animals and his contin-uous support. 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SNP/INDEL Discovery Pipeline based on CAP3 assembly[http://cgpdb.ucdavis.edu/SNP_Discovery/]Additional File 12Highly informative intragenic microsatellite markers. The table lists allelic data for a set of intragenic microsatellite markers, including the Contig ID, corresponding UniGene cluster ID, number of alleles, type of microsatellite. In bold case are figured contig/ESTs/UG clusters for which at least two alleles have a frequency higher than the mean (calculated as the total number of ESTs divided by the number of alleles) or higher than 33%. Alleles number and frequency are shown in bold if the frequency is higher than the mean or higher than 33%. A * is indicative of more than one repeat polymorphism observed for that cluster.Click here for file[http://www.biomedcentral.com/content/supplementary/1471-2164-8-118-S12.xls]

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RESEARCH ARTICLE Open Access

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