Current approaches for genome-wise functional analyses, such as microarray and RNA interference studies, rely on the specificity of oligonucleotide sequences to selectively target cellular transcripts. The design of specific oligos involves the determination of unique DNA regions in the gene/transcripts of interest from the targeted organism. This process is tedious, time consuming and it does not scale up for high-throughput studies.Taking advantage of the availability of complete genome sequence information for mouse and human, the most widely used systems for the study of mammalian genetics, we have built a database, [X]uniqMAP, that stores the precalculated unique regions for all transcripts of these two organisms. For each gene, the database discriminates between those unique regions that are shared by all transcripts and those exclusive to single transcripts. In addition, it also provides those unique regions that are shared between orthologous genes from the two organisms. The database is updated regularly to reflect changes in genome assemblies and gene builds.Over 85% of genes have unique regions at least 19 bases long, with the majority being unique over 60% of their lengths. 14482 human genes share exactly at least a unique region with mouse genes, though such regions are typically under 40 bases long. The full data are publicly accessible online both interactively and for download. They should facilitate (i) the design of probes, primers and siRNAs for both small- and large-scale projects; and (ii) the identification of regions for the design of oligos that could be re-used to target equivalent gene/transcripts from human and mouse. Following the completion of several whole genome sequencing projects a considerable effort has been focused on genome-wise functional analyses of a number of organisms (reviewed in ). Some oThe [X]uniqMAP database has been developed to store and present already pre-calculated unique regions for all EnsEMBL transcripts of the human and mouse genomes , the mos[X]uniqMAP is a collection of three databases: human and mouse uniqMAP as well as XuniqMAP. The first two collect the unique DNA 19-mers for all gene/transcripts within the two genomes. XuniqMAP collects those unique fragments within human and mouse that are also shared between them. The determination of the unique regions within a genome comprises three steps: (i) the construction of a set of non-redundant (NR) sequences, keeping track of the gene structure, from all the transcripts of each gene; (ii) self-comparison of the NR sequence set and storage of those positions found to be redundant; and (iii) deduction of the unique regions from regions identified in (ii) and gene structure data from (i).For each genome, the NR sequence set was established as follows. First, the central positions (i.e. the tenth nucleotide) for all possible 19-mers of all EnsEMBL transcripts, including non-coding RNAs and pseudogenes, were calculated. This length is around the standard minimum length for oligo design. All the central positions that fell within nine bases from the exonic boundaries were considered to be part of the regions that join exons whereas the others were part of the exon body  genes and 11350 (out of 11807) transcripts contained unique regions.For the calculation of unique positions shared between genomes, the two sets of unique fragments within the human and mouse genomes were compared against each other, storing information only for pairs of genes that shared at least five identical 19-mers in either sense. This arbitrary cut-off resulted almost completely in one-to-one, typically orthologous, relationships between human and mouse genes. The final set contains 15104 pairs made from 14482 and 14645 genes from human and mouse, respectively. As above, the gene/transcript relationships from each organism were transferred to the shared sequences which were then labelled according to their potential range of action across both organisms .A summary of the information held currently in the database, based on EnsEMBL release 40, can be found in Figure [X]uniqMAP contains a collection of the unique regions of all gene/transcripts in human and mouse as well as those unique sequences shared between their orthologs. For high throughput studies the complete data sets can be downloaded as FASTA files from the web site for further in-house calculations. The description lines of the sequences in the FASTA files contain all the appropriate information about the genomic co-ordinates and gene/transcript coverage of the fragments (see below for a more detailed explanation). On the other hand, in the more general case of experimentalists interested in only one or just few genes, this information can be retrieved interactively from the web site by searching the database either with an identifier or a nucleotide sequence.When a sequence is provided, this will be BLASTed  against Only gene and transcript Ensembl identifiers are valid when searching by an identifier. Once the identifier is provided, the database will recognise automatically to which organism it corresponds, therefore the organism option does not need to be specified unless to target directly the set of mouse-human shared unique regions. For each gene entry a web page will show all the information stored in the database by means of graphical and sequence displays. In uniqMAP, the graphical display is split into two levels, namely gene and transcript, corresponding to regions shared by all transcripts or by a single transcript, respectively Figure . In both>gene_id|transcript_id|symbol|chromosome_number:genomic_coordinates(strand)|fragment_lengthwhere the value of symbol can be either Ug or Ut, depending on whether the fragment is shared by all transcripts in a gene or unique to a single transcript, respectively. For the former case, gene_id and transcript_id will be the same and it will correspond to the EnsEMBL gene identifier.In XuniqMAP, the display is also split into two levels, namely reference and target, corresponding to all the unique regions of the gene in the reference organism and those matched by the target organism, respectively Figure . The col>target_gene_id:target_transcript_id:target_symbol|reference_gene_id:reference_transcript_id:reference_symbol|target_chromosome_number:targtet_genomic_coordinates(target_strand)|fragment_lengthwhere, as in uniqMAP, the symbol nomenclature will describe whether the fragment is shared or not by all transcripts.[X]uniqMAP is a collection of databases that stores all unique regions within human and mouse gene/transcripts as well as the sub-set of these unique regions that are identical between these two organisms. All the information is provided in the adequate genomic context for each gene, reflecting the relationships between their transcripts. This discrimination between regions shared by all transcripts of a gene from those exclusive to individual transcripts should be useful to determine the range of action desired when designing oligos. For oligo design, [X]uniqMAP information could be fed, if necessary, directly to downstream pipelines (i) to assess further the adequacy of some segments as, for example, RNAi or primers; or (ii) for extra filtering if lower similarity to other potential cross-reactive molecules as well as the precise location of the mismatched positions must be accounted for. [X]uniqMAP is based on EnsEMBL and is updated in parallel with EnsEMBL to reflect changes in genome assemblies or gene builds.. Enquiries on technical or data access issues can be mailed to webmaster@mitocheck.org.[X]uniqMAP is available online from RD conceived the initial idea about a database to store unique genomic regions. JLJ designed and implemented the data pipeline and database. JLJ and RD analysed the data and wrote the paper. Both authors read and approved the final manuscript.


The effectiveness of orthotic treatment continues to be controversial in international medical literature due to differences in the reported results and conclusions of various studies. Heterogeneity of the samples has been suggested as a reason for conflicting results. Besides the obvious theoretical differences between the brace concepts, the variability in the technical factors can also explain the contradictory results between same brace types. This paper will investigate the degree of variability among responses of scoliosis specialists from the Brace Study Ground of the International Society on Scoliosis Orthopedic and Rehabilitation Treatment SOSORT. Ultimately, this information could be a foundation for establishing a consensus and framework for future prospective controlled studies.A preliminary questionnaire on the topic of 'brace action' relative to the theory of three-dimensional scoliosis correction and brace treatment was developed and circulated to specialists interested in the conservative treatment of adolescent idiopathic scoliosis. A particular case was presented (main thoracic curve with minor lumbar). Several key points emerged and were used to develop a second questionnaire which was discussed and full filed after the SOSORT consensus meeting .Twenty-one questionnaires were completed. The Chêneau brace was the most frequently recommended. The importance of the three point system mechanism was stressed. Options about proper pad placement on the thoracic convexity were divided 50% for the pad reaching or involving the apical vertebra and 50% for the pad acting caudal to the apical vertebra. There was agreement about the direction of the vector force, 85% selecting a 'dorso lateral to ventro medial' direction but about the shape of the pad to produce such a force. Principles related to three-dimensional correction achieved high consensus (80%–85%), but suggested methods of correction were quite diverse.This study reveals that among participating SOSORT specialists there continues to be a strongly held and conflicting if not a contentious opinion regarding brace design and treatment. If the goal of a 'treatment consensus' is realistic and achievable, significantly more effort will be required to reconcile these differences. Orthotic bracing is the most common non-surgical treatment for Adolescent Idiopathic Scoliosis (AIS), either alone or in combination with exercises. In spite of some negative reports -3, braceGenerally speaking, bracing should unload the growth plates of the apical vertebral bodies on the concavity. Stokes has shown that an imposed vertebral deformity can be corrected by reversing the load used to create it . TherefoDesign principles for many braces are contradictory and based on dated concepts, proposed and tested decades ago when the three-dimensional (3D) nature of AIS was rarely considered or incorporated into the brace design. The proper biomechanical principles for orthotic correction should apply derotational forces that correct in the coronal and axial plane in addition to producing normal spinal alignment in the sagittal plane .In spite of numerous papers reporting on brace biomechanics -28, no pThis paper will investigate the degree of variability among responses of scoliosis specialists from the Brace Study of the International Society on Scoliosis Orthopedic and Rehabilitation Treatment -SOSORT- (described below) regarding brace action and the selection of force vectors to effect optimal curve correction. Each participant was given the same patient scenario and curve pattern and was asked to complete a questionnaire indicating their treatment approach. Ultimately this information could be a foundation for establishing a consensus and framework for future prospective controlled studies.The formation of the SOSORT was a significant accomplishment of the Conference on Conservative management of Spinal Deformities, held January 2004 in Barcelona, Spain . At thisHowever, there was obvious disagreement, between different schools of thought, about how braces should correct a particular scoliosis pattern. Intensive discussion on brace action was postponed to future meetings, as participants sought a consensus before designing a prospective controlled study.Following the formation of SOSORT in Barcelona, January 2004, a preliminary questionnaire was developed and circulated to all attendees and many specialists interested in the conservative treatment of adolescent idiopathic scoliosis. The simple questionnaire solicited their opinion on the topic of "brace action "where to push" and "why"" relative to the theory of 3-D scoliosis correction and brace treatment.Originally, two clinical cases were presented; only one was selected for use in this study. Complete Patient information including dorsal, ventral, lateral and forward bending photographs, as well as AP and lateral radiographs were provided Figure . While tTwenty-one questionnaires were completed and returned for inclusion in this study. All but two of the responses recommended using a TLSO in treating the specified case. Of the two other responses, one did not answer this question and the other selected a Milwaukee brace. The most consistent selection was The Chêneau brace (13 times) and its variations. In addition, the Lyon brace was chosen three times and the modified Boston was chosen once.The responses were almost evenly divided regarding the proper placement of the thoracic pad on the convexity of the thoracic curve. Eleven selected pad placement at the level of the apical vertebra and ten placed it below the apical vertebra but at the apical rib. Figure When asked about the importance of the 'three-point system' principle in the frontal plane, nearly everyone agreed that correcting the scoliotic curve in the frontal plane was a high priority figure . In the Addressing the necessity of a 'ventral to dorsal' force on the concave thoracic side by pushing with a pad on the ventral rib hump, fifty-six percent of the responses gave this principle high priority, 22% medium priority and 22% low priority Figure . The queReconstruction or normalization of the sagittal profile was considered an important concept Figure . All theWhen asked to identify which vector force they would use to correct the lumbar convexity, 76% of the group recommended a force reaching the apical vertebra of the lumbar curve Figure . There wWhen asked about the necessity for a ventral pad on the abdominal area, 37% percent of the experts did not recommend pushing ventrally figure . Ten perThe design of the pelvic section was the last area of focus. Figure The results of this study show the diversity of ideas and personal interpretations about the biomechanics of correction, brace design and treatment application in the proposed case. There was a wide range of treatment responses for this adolescent girl diagnosed with a classic idiopathic pattern of main right thoracic and minor left lumbar curvatures. Consensus was reached for three main biomechanical principles, at least from a theoretical point of view.First, nearly everyone agreed on the importance of using the 'three point system' to correct the main thoracic curvature in the frontal plane. Principles related to 3D correction like 'derotation' and 'normalization of the sagittal profile', achieved greater consensus after the meeting (80%–85%) rather than before (50%). However, the suggested methods of correction were quite diverse.When participants were asked which brace they preferred for this particular case, the Chêneau brace was the most frequently recommended. However, the variability in answers to specific questions on the biomechanics of correction indicates there are differing treatment methodologies. Consequently, the use of a particular name for a custom made brace, like the Chêneau, is no guaranty that there is a consistent standard in design or treatment.A high thoracic extension on the concave thoracic side acts as the upper oppositional counter pad of the 'three point system' in this case. However, its effectiveness is enhanced or minimized by the pad placement and the direction of the force vector pushing the thoracic convexity. Over-correction of the shoulder level by using a left high thoracic counter pad was recommended by 50% of respondents, while the other 50% preferred a neutral, balanced alignment with no overcorrection. Several biomechanical studies have supported the use of the" three point system". Jonasson-Rajala et al  and lateOpinions about proper pad placement on the thoracic convexity were evenly split. Fifty percent opted for the pad reaching or involving the apical vertebra and the other 50% for the pad acting caudal to the apical vertebra but including the apical rib. However, 85% of respondents agreed upon the direction of the force vector, selecting the 'dorso lateral to ventro medial' direction. Both mechanisms, the placement of the pad and the direction of the force vector are important for the 'three points system' principle as well as for 3D correction principles.The necessary combined forces to correct a thoracic scoliosis in 3D has been shown by Gignac D et al,  in a simIn this consensus study the direction of the force vector pushing the thoracic convexity, which has been recommended for the majority of the participants, includes the lateral to medial direction. However it also includes a dorsal to ventral force, which would reduce the thoracic kyphosis. Designing a ventral pad pushing ventral to dorsal on the concave thoracic side could prevent this. This pad, oriented close to the frontal plane, in combination with a secondary dorsal pad on the convex side, oriented close to the sagittal plane, would be the suggested 3D mechanism. It effectively creates a 'pair of forces' to derotate in the axial plane and a coupling mechanism to increase the thoracic kyphosis.Theoretically, this is the biomechanical principle of the Chêneau brace, the most recommended brace in this study. Despite this technical knowledge and preference for this specific brace, and though many of the specialists talk '3D correction', ironically, very few of them indicated an effective application of it. This could be the reason why the Chêneau brace has been reported to avoid thoracic flat back, for some authors ,31,32, wResponses to the questions about abdominal pressure and the design of the pelvic section show not only a lack of agreement but contradictory design principles. Perhaps indicating that these are not essential elements in stabilizing the curvature in the proposed case or are concepts requiring further exploration and discussion before agreement is reached.Although, all the SOSORT participants are recognized specialists in the treatment of scoliosis, having extensive knowledge of medical literature and significant clinical experience, many international opinions are missing from this study. Their inclusion would have increased the sample size and may have affected the outcome. Nevertheless, the objective of this study was not to establish new treatment guidelines as much as measure the level of consensus regarding current theoretical and treatment principles.Through open discussion and completion of specific questionnaires a 'treatment consensus' was sought; a foundational consensus, essential for creating a future, prospective, multicenter, multinational study on bracing effectiveness. While the interactive process and discussions were constructive and some points of agreement were identified...was there sufficient progress towards an essential consensus?. Unfortunately, this study reveals that among participating SOSORT specialists there continues to be a strongly held and conflicting if not contentious opinion regarding TLSO design and treatment. If the goal of a 'treatment consensus' is realistic and achievable, significantly effort will be required to reconcile these differences.The author(s) declare that they have no competing interests.MR, SN, HRW, TBG, TM, TK conceived of the study, and participated in its design and coordination and helped to draft the manuscript.SN also performed the statistical analysis.


One of the most powerful methods for the prediction of protein structure from sequence information alone is the iterative construction of profile-type models. Because profiles are built from sequence alignments, the sequences included in the alignment and the method used to align them will be important to the sensitivity of the resulting profile. The inclusion of highly diverse sequences will presumably produce a more powerful profile, but distantly related sequences can be difficult to align accurately using only sequence information. Therefore, it would be expected that the use of protein structure alignments to improve the selection and alignment of diverse sequence homologs might yield improved profiles. However, the actual utility of such an approach has remained unclear.We explored several iterative protocols for the generation of profile hidden Markov models. These protocols were tailored to allow the inclusion of protein structure alignments in the process, and were used for large-scale creation and benchmarking of structure alignment-enhanced models. We found that models using structure alignments did not provide an overall improvement over sequence-only models for superfamily-level structure predictions. However, the results also revealed that the structure alignment-enhanced models were complimentary to the sequence-only models, particularly at the edge of the "twilight zone". When the two sets of models were combined, they provided improved results over sequence-only models alone. In addition, we found that the beneficial effects of the structure alignment-enhanced models could not be realized if the structure-based alignments were replaced with sequence-based alignments. Our experiments with different iterative protocols for sequence-only models also suggested that simple protocol modifications were unable to yield equivalent improvements to those provided by the structure alignment-enhanced models. Finally, we found that models using structure alignments provided fold-level structure assignments that were superior to those produced by sequence-only models.When attempting to predict the structure of remote homologs, we advocate a combined approach in which both traditional models and models incorporating structure alignments are used. The current stream of genome sequence data has lead to a bottleneck between DNA sequencing and elucidation of protein function . TherefoHomology-based methods for structure prediction rely on the observation that proteins that share a common ancestor will usually have similar sequences, and that, in turn, similar protein sequences produce proteins with similar fold and (often) similar functions . HoweverThe profile-type methods address this problem by incorporating family-specific information inherent in multiple sequence alignments into sequence searches -15, and Because profile HMMs model the information present in a sequence alignment, they are affected by quality of the input alignment , but theGiven the limitations inherent in sequence alignments as inputs for profile HMMs, researchers have explored the use of sequence alignments derived directly from structural alignments of proteins, particularly in cases where superfamily-level assignments are desired ,26-31. SOne limitation in using structure alignments for profile HMMs is the weak level of structure representation in many superfamilies. In addition, coverage of a given superfamily is often strong in one area and weak in others. Hence, models made from alignments of these sequences, even if supplemented with additional homologous sequence, may not optimally describe the entire superfamily in question. It has also been suggested that there is an optimal range of sequence similarity for training of profile HMMs, and that using a very broad range of diverse sequence can lead to "profile dilution", reducing model quality ,32.et al. and Sillitoe et al. have suggested that pools of models, each built iteratively from a single structural representative (referred to here as the "master" sequence) provide better performance at the superfamily level than models incorporating structure alignments of multiple masters [et al. also reported that combining the two types of models yielded essentially no benefit [Accordingly, the literature record for the use of structure alignments for profile HMMs yields mixed results. Several researchers, using a variety of experimental arrangements, have reported that HMMs based on structure alignments do not provide a benefit over standard HMMs. Gough  masters ,30. Sill benefit . Howeveret al. used structure alignments to augment iterative profile construction, and reported improved results when attempting to predict very distant homologous relationships. However, some of the improvement reported was based on the combination of the structure alignments with additional structure-derived information (secondary structure and solvent accessibility) [et al. explored the inclusion of structure alignments in the construction of profiles in a profile-to-profile approach based on PSI-BLAST [et al., they found that profiles utilizing structure alignments provided some improvement over standard models, and that this increase in performance could be furthered through the addition of other structural data into the models. Casbon and Saqi tested a profile-to-profile approach relying on a hybrid PSI-BLAST/HMM protocol [vice versa. This suggests that the two types of models might be complementary if used in combined searches.Other researchers have provided qualified support for the use of structure alignments in the construction of profiles  . Other gSI-BLAST . Similarprotocol . They foWe undertook a large-scale assessment of the utility of structure alignments for the generation of profile HMMs, using the traditional sequence-to-profile method. First, we tested several iterative protocols to determine which method generated the most sensitive profile HMMs with HMMER , using oIn order to determine the best iterative methodology for building both single-master HMMs and SLAHMMs with HMMER, performance was compared for four different parameter sets . In all cases, an alignment was built through repetitive HMM searches against a sequence database (with re-training of the HMM after each cycle), but the parameter sets tested different cutoffs for sequence inclusion into the growing alignment, use of heuristics to improve the alignment, and the method for aligning sequences to the model to create the alignment. Test sequences (or "probes") were then searched against the resulting models and the correct or incorrect structure assignments recorded. The superfamily assignments provided in SCOP ,34 were When single-master HMMs were compared to each other for all four parameter sets PS1-PS4), the difference in performance was extremely small  also resulted in relatively similar performance between the various iterative parameters Figure . HoweverHowever, the results also suggest that the use of the heuristics (to select sequences for addition to the model) applied in PS1 and PS2 had a beneficial effect on the quality of the models. Both PS1 and PS2 outperformed counterparts that did not use the heuristics. In addition to the small performance improvement, the heuristics provided the practical benefit of smaller alignment sizes (because they are more selective with respect to added sequences), and therefore lower computational overhead.It was possible to use both sets of models together in a combined search, simply by placing both in a single database and searching the probes against this database. When performance of the combined models was compared for each parameter set, the difference again narrowed, much as it did for the single-master HMMs Figure . AlthougWhen directly compared, single-master HMMs clearly outperformed SLAHMMs Figure . This reAssessment of the specific probe assignments made by SLAHMMs vs. single-master HMMs confirmed that SLAHMMs made unique assignments. At a strict cutoff of 80 incorrect assignments (EPQ ~0.05), the two types of HMMs shared 839 correct assignments of the same probes . However, while single-master HMMs correctly assigned an additional set of 224 probes uniquely, SLAHMMs also correctly assigned 42 probes uniquely  were made prior to the first incorrect assignment. The rest were made after errors had been recorded. In contrast, of the unique assignments made by the single-master HMMs, 91 (~41%) were made prior to the first recorded error. This suggests that, as expected, SLAHMMs are very generalized models that capture more difficult assignments made right at the edge of "the twilight zone", but can miss easier matches.If the single master HMMs are allowed a looser cutoff of up to 1575 errors , they will correctly assign 31 of the 42 sequences that SLAHMMs capture at a cutoff of 80  but did not actually require the explicit incorporation of structural information in the form of a structure-based alignment. Further, it has been suggested that highly accurate alignments are not essential to the production of useful profile HMMs .To see if structural alignments were required, we aligned our test protein domain sequences with ClustalW . These sTo make the iterative HMM searches computationally tractable during initial model building, BLAST was used to pre-filter a large pool of possible homologs from the sequence database for each SCOP master domain (see methods). Though it provides practical benefits, the BLAST pre-filter could also limit the possible sequence space available for training each single-master HMM. Thus, it could be argued that the primary benefit of SLAHMMs might be primarily practical rather than theoretical: they allow the HMM to sample a broader range of sequence space, without the computationally intensive requirement of iterating directly against the entire sequence database.To help determine the practical vs. theoretical benefits of SLAHMMs, the 42 probes that SLAHMMs uniquely assigned at an EPQ ~0.05 , making the additional HMM training requirements minimal. Since the alignments used to build SLAHMMs are derived from sequence alignments already available from the generation of standard, single-master HMMs, they can be conveniently created as an adjunct set of models to improve overall performance.The primary limitation of SLAHMMs is that they require at least two structural representatives per superfamily to be modeled, and some superfamilies still lack even a single structural representative. However, with the gradual growth in structural data likely to be generated from the various structural genomics projects ,39, mult-3. The intent of the chosen E-value cutoff was to provide a balance between broad coverage of the resulting models  and challenging potential assignments with which to test the methods. The chains included the ASTRAL "genetic domains", and were filtered to be at least 80 residues in length. Domains from only the first five classes in the SCOP hierarchy were used, as these represent the "typical" globular proteins to which this work aims to successfully assign structures.The SCOP structural classification database ,34 has bThese domains were used both for model building and benchmarking. The filtered ASTRAL set from above was additionally filtered to only retain superfamilies with three or more members, so that models could be constructed based on the structure alignment of two sequences, while still retaining a sequence to test the resulting model (leave-one-out test).The selected SCOP/ASTRAL superfamily representatives are distant homologs, and it is difficult to obtain an accurate alignments of them using sequence information alone -25. Ther17 structures and one superfamily were removed from the set because of problems caused by inconsistencies between the ASTRAL sequence records and their corresponding PDB files. Also, our CE variant was in still in early development, and failed to align 14 superfamilies. Therefore, these were also removed from the test set. These removals reduced the set of initial structures from 257 superfamilies/1995 chains to 242 superfamiles/1575 chains. However, all five classes of SCOP were still well represented. A breakdown of the representation of SCOP fold classes in this sequence set is avaliable in The Genbank non-redundant protein sequence database ("nrprot") was utilized for the collection of sequence homologous to the SCOP domains. Two releases were used, an older release (downloaded 10/18/02) for early development and testing of the method . All BLAST hits to each SCOP domain with an E-value of < 500 were stored in a miniature database as possible homologs to that domain. HMM searches against the entire nr60 database were prohibitively slow, particularly for purposes of iteration, so pre-filtering the database was required. This technique was first described for the SAM-T98 protocol .hmmcalibrate; this provided additional scoring accuracy. Reported E-values were based on the default theoretical background database size in HMMER . We fouwissprot  was usedall of the collected sequences were aligned back to the model, including the SCOP domain. The domains collected from BLAST can be somewhat shorter than ideal for representing the sequence space around the SCOP domain (even with filtration for coverage), and the aim of this alignment step was to lengthen the model such that it encompassed all conserved sections of the SCOP domain.The ClustalW-created sequence alignment was used to train an initial HMM with HMMER. This model was then used to search the pre-filtered sequence database (matching the SCOP domain) for additional homologous sequences. This model was built as a global/local searching model, meaning that it would attempt to capture sequences that span the length of the model. In the first iteration, After the first iteration was complete, the alignment produced was again used to build and calibrate another HMM using HMMER. The pre-filtered database was again searched and the sequences scoring below a set E-value threshold (determined by the parameter set as discussed below) were collected and aligned back to the model. The model was then re-built using this new alignment, and another iterative cycle initiated, etc.We tested 4 different iterative strategies in order to determine which would be the most successful at later structure assignment Table . We nameFrom the second iteration onward in PS1, only the newly collected sequences were freshly aligned to the model, while the prior alignment was retained unchanged. This arrangement allowed sequences at each level of similarity to align to a model most suited to their relationship to the SCOP domain. It also guarded against iteration and alignment drift by anchoring both the alignment and the model to the initial SCOP domain.The iteration was run for a total of six cycles. With each cycle, the E-value threshold was raised to allow more distant sequences into the alignment as detailed in Table In addition to the E-value criterion, sequences were filtered prior to addition to the alignment at each iteration based on heuristics that aimed to improve the quality and relevance of the alignments. First, sequences were filtered based on their coverage of the HMM model, calculated as the percentage of match states available in the model aligned to by the sequence. It was determined empirically that a reasonable cutoff level was 70% model coverage in the first three iterations and 50% model coverage in the last three iterations. Sequences that aligned to the model below these cutoffs were almost always short fragments or poor alignments.In addition to the filtration for coverage, a heuristic was applied to screen out poor alignments. The measure used was the ratio:is is the number of insertion states in the model caused by the sequence and ms is the match states the sequence matched to. The point of this measure was to detect poor alignments in which a sequence only matched a few highly conserved match states, but otherwise aligned to insert states in the model . These sorts of alignments were sometimes seen at the higher E-value  thresholds, and were presumably not helpful to the construction of a good model. Through the use of Rim, large insertions in the model were still possible, provided the sequence also aligned to a large amount of match states. This prevented the exclusion of sequences simply because they produced a large but valid insertion in the model. Rim was required to be less than 10 for the initial iteration and less than 3 for all other iterations. Rim was set to a very high level in the first iteration because alignments at the low E-value used will almost always provide a very good alignment without any heuristic.where Rim progression, but continually re-aligned all sequences back to the new model after every iteration. PS3 and PS4 tested a much less controlled iteration, in which the heuristic parameters were essentially turned off, as well as a more rapid E-value progression .At the end of the iterative cycle each SCOP domain had a matching sequence alignment corresponding to the sequence space surrounding it, as well as a profile HMM that provided a model of the alignment for purposes of sequence searches. At this point, the model was stored as a single-master HMM for later use in the benchmarking experiment, while the sequence alignment was used in the creation of SLAHMMS.Structure alignments were used to generate a high-quality multiple sequence alignment of the SCOP master domains for each superfamily. The sequence alignments to each SCOP master were then grafted on to the structure alignment scaffold by using the one-to-one correspondence of the SCOP master sequence in both alignments. In most cases, the SCOP master did not participate in some columns of the sequence alignment; these columns were removed prior to the merging procedure. In large superfamilies, the resulting alignment could contain thousands of sequences.The superfamily alignments generated by this merging process often had redundant copies of sequences from the nr60 database. These redundancies occurred because several SCOP masters detected and incorporated the same sequences as they iteratively build alignments. This was expected and desirable, as it meant the HMMs were reaching far into the available sequence space. The redundant sequences were removed such that only the best instance of each sequence was retained.The best instance of a sequence in an alignment was chosen based on its relationship to the SCOP master that retrieved it during the initial iterative alignment construction. The aim was to select the instance that was likely to be in the highest quality alignment with its SCOP master. This was determined using a set of cascading tests using the data stored during the iteration runs. In order, the tests were: iteration cycle at which the sequence was added to its alignment, the E-value it matched the model with at the time of its addition, and the length of the sequence fragment (a longer length implies a better SCOP master partner for the sequence). The sequence instance which scored best in these tests was retained in the superfamily alignment, and all other instances of the same sequence were removed. Once the redundancy had been removed from the alignment, it was suitable for submission to HMMER to generate a SLAHMM representative of the entire superfamily.prior to the redundancy removal described above, so that the maximum number of sequences collected without the use of the removed domain could be retained. The net effect of the procedure was to create alignments that essentially were constructed as if the removed SCOP master was never present. The SLAHMMs could then be directly compared with the single-master HMMs.In normal use, it is expected that a single SLAHMM model would be generated and stored. However, for purposes of benchmarking it was necessary to remove a SCOP master and all of the sequence it detected from the alignment, so that that domain could be tested against the (now uncontaminated) model in a leave-one-out test. A version of the alignment for each SCOP master was generated, which was missing that master and its detected sequences. This was done a posteriori method for match state generation [hmmbuild. The use of 25 match states as a cutoff was arbitrary, but seemed reasonable based on reviews of the alignments.When producing SLAHMMs with HMMER, difficulties were encountered in a few superfamilies. It appeared that the cause was the input alignments, as these problems were not encountered with the single-master alignments. In some superfamilies, the superfamily-wide alignments were very sparse, with large sections filled mostly with gaps, and areas of conserved structure with little or no recognizable sequence conservation. This type of alignment occasionally caused severe model shortening with HMMER where, despite the input of a large alignment, HMMER would generate a model containing less than 25 match states. In such cases, this problem was addressed by turning off the more sophisticated maximum neration  and usinhmmbuild for three alignments in both PS3 and PS4. Therefore, in order to provide PS3 and PS4 with the same amount of models as PS1 and PS2, models from PS1 and PS2 were copied over to their closest partners  for these cases. In three cases, the models did not make any assignments in the subsequent benchmarking studies. Thus, the added models should have no notable effect on the results for PS3 and PS4. The net result of the above construction steps was 1575 single-master HMMs and 1575 SLAHMMs  for each parameter set.HMMER crashed when attempting to build a model with During collection of the final results Figures , 3, 4, Hone possible correct SLAHMM hit . Probe-centric scoring allowed for direct comparison of the number of correct assignments between the two methods. All incorrect assignments for a given probe where counted when determining coverage vs. error [The experiments were run by using each SCOP master sequence as a probe against all of the models of each type, as well as a combined database containing all the models. Structure assignments were scored in a "probe-centric" fashion, in which each SCOP domain could only be correctly assigned once. This was done because for each of the 1575 domains, there were at least two correct superfamily-level hits available in the case of single-master HMMs, but only s. error ,40, withdifferent superfamily but the same fold were counted as correct hits, and matches of a probe to a model in the same superfamily were ignored . The requirement for a cross-superfamily match meant that some of the probes from the superfamily tests could not possibly be correctly assigned in the experiments (because no models were available from another superfamily in the same fold); these were removed, leaving 675 probes (and possible correct assignments). To expand the size of the test set, we added additional probes from smaller superfamilies which had not been used to make any models because they did not have sufficient structural representatives to meet our initial superfamily filtration criteria. Inclusion of these additional test sequences increased the size of the test set substantially, from 675 sequences from 88 superfamilies to 933 sequences from 291 superfamilies. Thus, the reported theoretical EPQ values for fold recognition .Underpredictions refer to cases where the SCOP authors did not group similar structures into the same fold grouping, even though structural comparison indicates a similar overall fold (and perhaps even an evolutionary relationship). Hence, the structures are grouped only into the same vice versa). In both directions, an E-value less than 1 was required. Second, the structures providing the sequence match (both from a model and probe standpoint) were required to align with a Z-score of 4 or greater using CE [To determine which fold-level pairings would be provided such special treatment, we adopted a standard based on both sequence and structural characteristics. First, the HMM match between domains at the sequence level was required to be bi-directional . It seems likely that the regular α/β alternation in these domains produced a distinctive generalized sequence signal that our HMMs detected. It is unclear if these matches were simply the result of analogy or a true homologous relationship. To our knowledge, detectable sequence similarity between these folds has not been previously reported.Artifact generation refers to cases where, because of a SCOP domain's architecture, models built using that SCOP domain incorporated sequences related to other SCOP domains. Then, when the experiment was run, these models correctly recognized these other SCOP domains, but this recognition was treated as an "error" because these domains were not part of the same SCOP superfamily as the domain used to initiate the model.Specifically, such artifact generation occurred where a protein consisted of two interacting fragments that flank a central fragment. In some cases, SCOP split such a protein into two compact domains, with one domain consisting of the central fragment, and the other consisting of the two flanking regions joined together at the break points into a single "composite domain". When this flanking domain was used to build an initial ClustalW alignment in the protocol described above, the central fragment was incorporated via the homologous sequences brought into the initial alignment . The resulting models then recognized the central fragment as a homolog.Only two superfamily pairings displayed these types of errors for multiple domain and model matches. In one pair, the domains of b.92.1  flank those of c.1.9 . In the other, some  domains of c.4.1 (nucleotide binding domain) flank those of c.3.1 (FAD/NAD(P)-binding domain). These matches were therefore ignored in the collection of the results.Cases involving other superfamilies were also detected, but in every one of these cases, the errors did not occur for multiple probes and models across the superfamily. They only occurred for a probe and its corresponding flanking domains with the same PDB ID and chain designation. Therefore, all of these cases could easily be dealt with by adding a simple prohibition to the collection of results: any errors where a probe and matching model shared the both the same PDB ID and chain were ignored. This prohibition automatically removed most artifactual matches, while having a minimal effect on the collection of legitimate errors .superfamilies scored higher?Although the methods above removed all obvious cases where correct hits were mislabeled as errors, it was important to institute a generalized method to help suppress the effect of these sorts of errors, in the event that some were missed. This was done by limiting the amount of errors counted for a given probe to one per incorrect superfamily match. This had the effect of allowing artifacts and underpredictions to only add one incorrect match to the total for each probe, as opposed to several. At the same time, it would be expected that limiting counted errors in this fashion would only have a small effect on the counting of legitimate errors, as these sorts of errors tend to be random and so come from multiple different superfamilies for a given probe. As described above, only the first correct match of probe to its superfamily was counted. By only counting each incorrect superfamily once per probe, the experiment effectively asked the question: at a given score cutoff, has this probe been assigned to the correct superfamily, and how many incorrect ES conceived of the study, performed the experiments and drafted the manuscript. PB helped conceive the study and edited the manuscript. Both authors read and approved the final manuscript.Click here for file


Patients in the intensive care unit (ICU) may be in an inadequate condition to give their informed consent for research. The aim of this study was to analyse the ability to recall participation in a clinical trial for which ICU patients had given their consent.The data presented are a two-step observational study: first, a protocolled informed consent procedure was conducted then the informed consent was given by the patient, and second, a patient interview was held 10 ± 2 days later by the same investigator. The primary endpoints were the ability to recall their participation in the clinical trial, as well as its purpose and related risks. As secondary endpoints, we investigated whether asking questions about the clinical trial or reading the informative leaflet was related to the recall. To be included in the study, the patient had to have a Glasgow Coma Scale score of 15, be fully oriented and free of mechanical ventilation, and be judged competent by both the investigator and the attending physician. Patients admitted to the ICU after major surgery or trauma were eligible. However, patients who refused to participate, or those whose next-of-kin gave consent, were excluded.Of the 44 patients, 35 (80%) recognized, 10 to 12 days after informed consent had been obtained, that they had participated in the clinical trial, but only 14 out of 44 (32%) could recall the clinical trial purpose and its related risks. More patients with complete recall had read the informative leaflet or asked at least one question before signing the informed consent. Asking at least one question was associated with complete recall.Our results confirm that obtaining informed consent for research during an ICU stay is associated with poor patient recall of participation in a clinical trial and its components (purpose and risk). Whether encouraging reading the informative leaflet and asking questions about the clinical trial improves the informed consent procedure remains to be fully investigated. Within the past few decades, medicine, especially critical care medicine, has progressed spectacularly as a result of medical research and the participation of patients in clinical trials. The Declaration of Helsinki and international guidelines require that the patient give informed consent before participating in a study. This informed consent is essential to respect the autonomy of patients and protect them against abuse -3.Therefore, to respect the ethical principles of clinical research, informed consent for research demands to be held at a high standard because the patient does not always benefit directly from the research results and might suffer some risks for the good of the community. Keeping with this high standard, informed consent must fulfil three mandatory conditions as described in the Belmont report: adequate information about the study with a complete disclosure of the risks and benefits, the patient's comprehension, and the voluntariness -7. This This investigation on informed consent was an observational substudy of a clinical trial about inflammation, described elsewhere . The preThis study was performed in a 20-bed surgical ICU of a tertiary-university-affiliated teaching hospital receiving 1,600 patients per year.Patients admitted to the ICU after a major surgery or trauma were eligible. To give their consent to participate in the clinical trial on inflammation, patients had to present with a GCS score of 15, be fully oriented and free of mechanical ventilation, and judged competent by both the investigator and the attending physician. Patients who refused to participate, met any of the exclusion criteria for the clinical trial on inflammation, and those whose next-of-kin gave consent were not considered for the present study. Furthermore, incompetent or non-French-speaking patients, and those with psychiatric disorders, senile dementia or other intellectual disabilities were not included.A protocoled procedure detailing how to obtain informed consent was developed to ensure that the two investigators contributing to the study obtained consent in an identical manner. The investigators were physicians who were not caring for the patients enrolled in the study. Informed consent was obtained on the ICU admittance date, after a 20-minute individual oral presentation. During this presentation, the investigator explained the clinical trial on inflammation, emphasizing two clinical trial components: the purpose and its related risks. The defined keyword for the clinical trial purpose was inflammation. The clinical trial risk for the patient was that 10 ml of their blood would be drawn daily for the first five days of their ICU stay, and a final blood draw would be required at day 28. The investigator told the patient that the risk was minimal.a priori in the consent procedure. No other attitudes of the patient were recorded or tested.The patient then received a one-page informative leaflet. At the end of this procedure, the investigator asked the patient if he had any questions about the clinical trial before signing the informed consent form. The investigator noted whether the patient asked any questions and/or if he read the informative leaflet in his presence. These two attitudes of the patient (asking one or more questions and reading the informative leaflet) were defined This informed consent procedure conforms to recommendations on the ethical conduct of clinical research involving patients in the ICU .Patient age, gender, history of daily alcohol intake type of admission and diagnosis, as well as Simplified Acute Physiological Score second version (SAPS II)  were recWhen informed consent was obtained, clinical and laboratory values, the Sequential Organ Failure Assessment (SOFA) score , and medAt 10 ± 2 days (range), the investigator met the patient to plan the blood sampling to be performed on day 28. At this time, the investigator assessed whether the patient could recall participation in the clinical trial and/or any of the clinical trial components.The primary endpoints were the ability of the patient to recall participation in the clinical trial, as well as the purpose and related risks of the clinical trial. As secondary endpoints, we investigated whether asking questions about the clinical trial or reading the informative leaflet was related to the recall.Patients who could report their participation in the clinical trial on inflammation, the clinical trial purpose and the related risks were assigned to the 'complete recall' group. Patients lacking one or more components were assigned to the 'incomplete recall' group.The clinical trial on inflammation itself, as well as the informative leaflet and the consent form, were approved by the Ethical Committee for Human Research of our institution. Specific informed consent had not been sought for this study. The educational status of the patient, that was the sole data not available in the dataset of the clinical trial, was subsequently retrieved from the administrative file.t test or a Mann–Whitney U test, as deemed appropriate. We also assessed the sensitivity, specificity, the positive predictive value, the negative predictive value and the likelihood ratio of factors that were statistically significant on the basis of the univariate analysis performed to predict complete recall of the clinical trial. Odds ratios with 95% confidence intervals were calculated to estimate the effect size of risk factors associated with complete recall. All tests were two-tailed; p < 0.05 was considered significant.StatView for Windows version 5.0.1  was used for statistical analysis. Patients were stratified as having either complete or incomplete recall; these data were compared separately by using a two-tailed Fisher's exact test, an unpaired Between May 2000 and January 2001 we included 48 patients, of whom four died during the ICU stay  recognized they had participated in a clinical trial; 14 of the 44 patients (32%) could recall their clinical trial participation as well as the clinical trial components, namely the clinical trial purpose and the related risk . Asking at least one question was associated with a complete recall of the clinical trial (p = 0.03). The sensitivity, specificity, positive predictive values, and negative predictive values of 'asking one question' and 'reading the informative leaflet or asking one question' to differentiate patients with complete recall from patients with incomplete recall are shown in Table When informed consent was obtained, the clinical and laboratory values of the patients did not differ between the two groups Table . The medThe first investigator included 15 34%) patients in the clinical trial during the first three months of the inclusion period, and the second investigator added 29 (66%) patients during the last six months. Patient characteristics, attitude during the informed consent procedure, and rates of recall did not differ significantly between investigators  in patients informed after being admitted to the ICU is very low. It is presumably much lower than in patients who gave their informed consent outside the ICU setting.• More patients who could recall their clinical trial participation and the clinical trial components had read the informative leaflet or had asked at least one question before signing the informed consent.• Reconsidering the informed consent procedure repeatedly during an ongoing clinical trial could be useful to respect patients' rights.• Informed consent to participate in a clinical trial should be considered an ongoing process rather than a single procedure.GCS = Glasgow Coma Scale; ICU = intensive care unit.The authors declare that they have no competing interests.CC participated in the design of the study and the collection, analysis and interpretation of the data and wrote the manuscript. PM contributed to the conception and design of the study and to the analysis and revision of the manuscript. SL was involved in the design of the study and the collection of data. BR conceived and coordinated the study and was involved in the interpretation of data and in revision of the manuscript. All authors read and approved the final manuscript.


A hierarchy of stability exists among the types of surgical movements that are possible with orthognathic surgery. This report updates the hierarchy, focusing on comparison of the stability of procedures when rigid fixation is used. Two procedures not previously placed in the hierarchy now are included: correction of asymmetry is stable with rigid fixation and repositioning of the chin also is very stable. During the first post-surgical year, surgical movements in patients treated for Class II/long face problems tend to be more stable than those treated for Class III problems. Clinically relevant changes (more than 2 mm) occur in a surprisingly large percentage of orthognathic surgery patients from one to five years post-treatment, after surgical healing is complete. During the first post-surgical year, patients treated for Class II/long face problems are more stable than those treated for Class III problems; from one to five years post-treatment, some patients in both groups experience skeletal change, but the Class III patients then are more stable than the Class II/long face patients. Fewer patients exhibit long-term changes in the dental occlusion than skeletal changes, because the dentition usually adapts to the skeletal change. A research grant focused on the outcomes of orthognathic surgery at UNC, funded by the National Institute of Dental and Craniofacial Research, enters its 28This research project has resulted in more than 100 research papers in peer-reviewed journals, and about half that many invited contributions and book chapters. It became obvious by the 1990s that a major influence on the outcomes of orthognathic surgery was the amount and direction of surgical movement. A series of research papers that focused specifically on stability as related to the different surgical movements was summarized in 1996 in a paper outlining a hierarchy of stability related to surgical movements . Th. Th29]. Problematic stability in moving the maxilla down is due largely to changes within the first few postsurgical weeks, before bone healing is complete, as occlusal force tends to push it upward Figure . There aWidening the maxilla with a segmental osteotomy stretches the palatal soft tissues, and this tissue elasticity provides a force to decrease the expansion post-surgically  changes are more likely in Class II/long face patients than in Class III patients. Fewer patients exhibit long-term changes in the dental occlusion than skeletal changes, because adaptive changes often occur in the dentition as skeletal changes occur. In both the post-surgical and post-treatment periods, almost all the changes occur in a minority of patients, so it is better to consider the percentage of patients with clinically significant changes than the mean changes. The database makes it clear that clinically satisfactory results can be obtained and maintained long-term in the great majority of orthognathic surgery patients, but the differences among various directions of movement must be taken into account when treatment is planned.The author(s) declare that they have no competing interests.Dr. Proffit served as principal investigator on the research grant that supported this work, and prepared the first draft of the manuscript. Dr. Phillips supervised the development of the project's data base, and was responsible for all statistical analyses. Dr. Turvey performed almost all the surgery for these patients and played a major role in gathering the clinical data on short- and long-term recalls. All three authors were involved in revision and final preparation of the manuscript.


The German cDNA Consortium has been cloning full length cDNAs and continued with their exploitation in protein localization experiments and cellular assays. However, the efficient use of large cDNA resources requires the development of strategies that are capable of a speedy selection of truly useful cDNAs from biological and experimental noise. To this end we have developed a new high-throughput analysis tool, CAFTAN, which simplifies these efforts and thus fills the gap between large-scale cDNA collections and their systematic annotation and application in functional genomics.CAFTAN is built around the mapping of cDNAs to the genome assembly, and the subsequent analysis of their genomic context. It uses sequence features like the presence and type of PolyA signals, inner and flanking repeats, the GC-content, splice site types, etc. All these features are evaluated in individual tests and classify cDNAs according to their sequence quality and likelihood to have been generated from fully processed mRNAs. Additionally, CAFTAN compares the coordinates of mapped cDNAs with the genomic coordinates of reference sets from public available resources . This provides detailed information about overlapping exons and the structural classification of cDNAs with respect to the reference set of splice variants.The evaluation of CAFTAN showed that is able to correctly classify more than 85% of 5950 selected "known protein-coding" VEGA cDNAs as high quality multi- or single-exon. It identified as good 80.6 % of the single exon cDNAs and 85 % of the multiple exon cDNAs.The program is written in Perl and in a modular way, allowing the adoption of this strategy to other tasks like EST-annotation, or to extend it by adding new classification rules and new organism databases as they become available. We think that it is a very useful program for the annotation and research of unfinished genomes.CAFTAN is a high-throughput sequence analysis tool, which performs a fast and reliable quality prediction of cDNAs. Several thousands of cDNAs can be analyzed in a short time, giving the curator/scientist a first quick overview about the quality and the already existing annotation of a set of cDNAs. It supports the rejection of low quality cDNAs and helps in the selection of likely novel splice variants, and/or completely novel transcripts for new experiments. The availability of a growing number of draft or completed genomes has shifted the most pressing challenges towards the understanding of the molecular and cellular biology of genes and other encoded elements. This understanding is facilitated by the interpretation of genomes not only in terms of their structure but also of their function and diversity. To this end cDNAs from several human large-scale sequencing projects -3 have pThe German cDNA Consortium has pioneered the large-scale sequencing of full-length cDNAs  and theiWe have developed a new high-throughput cDNA analysis tool, CAFTAN, which filters sequences based on their potential to be derived from full-length and fully processed transcripts and spliced forms. It identifies and filters those cDNAs containing incompletely processed or truncated transcripts, which are consequences of erroneous mRNA processing  or of erThe main strategy of CAFTAN is based on the mapping of cDNAs to the genome assembly, and the analysis of their genomic context. The presence and type of polyA signals, internal and flanking repeats, the GC-content, and splice site types are evaluated in different tests and aid in the classification of cDNAs into several groups according to their sequence quality. CAFTAN compares the coordinates of cDNAs in the respective genomic locus with the coordinates of a reference set of cDNA-sequences from publicly available resources  [see Additional files 1) input or raw data, 2) Extraction of simple and composite features from raw data, 3) Rules definitions, 4) Prediction and Evaluation, 5) Program specificationsThe implementation of the CAFTAN method can be summarized in the following stages: CAFTAN takes as input a multiple FastA file of the cDNAs to be analyzed and a BLAT output from the FastA sequence file in pSL format with header ,24.Simple and composite features were extracted from a training set of 3500 manually curated sequences from the German Human cDNA Project -DKFZ collection (supplementary information). Afterwards, the distribution of these features was studied depending on their tag- in the curated DKFZ set- , see section 2.2.The first step is to know where and how good the cDNAs are localized in the genome. For that purpose we use BLAT, which maps most cDNAs to a single position/locus in the human genome. However, recent gene duplication events, repeat elements in cDNAs, or misassemble of genome sequence contigs give rise to the mapping of cDNAs at multiple loci, making a post processing of the BLAT output (given by the user) necessary. Only the hit with the lowest number of mismatches is selected. The mapping positions of the selected hit are kept for use of this cDNA in further analyses.Afterwards, the distances between consecutive blocks- matches of the cDNA to the genome in the BLAT output- are analyzed; a block can be interpreted as an exon. The blocks are joined when the distance is less than 10 bp in the genome sequence. The final number of blocks and their exact genomic positions are recalculated and stored. This process is repeated for every cDNA.Additionally information on the number of mismatches, successfully mapped length, number of Ns, gaps etc. are stored for the later generation of composite features and rule definitions. Figure , Table 1PolyA sites in mRNAs are defined by a hexameric polyadenylation signal, AAUAAA, or a one-base variant thereof . This siWe have developed the program "Polyasignal", using EMBOSS libraries [see Additional files The user can define almost all variables, such as maximum distance allowed between the signal and the polyadenylation site (default 50nt), and patterns to match the polyA signals and polyA tail. Parameters are by default optimized for the human genome ,28 but iMost exon/intron boundaries match consensus dinucleotides that are specific for splice donor and splice acceptor sites at the 5' and 3' ends of exons, respectively . All putSplice Site Type is classified as canonical only when all of its splice sites are canonical, for other cases see Table Once the splice type of each intron in a cDNA has been defined, the Ss type of the whole cDNA is then defined Table . The cDNThe genomic sequence is further checked for CpG islands up to 200 bp from the beginning of the cDNA. We use the generally accepted definition of what constitutes a CpG island .Additionally, the presence of a stretch of multiple A's (polyA tail) in the genome at the matching position of the cDNA's end is checked. We look for a window of 20 bp in the genome taking the last 5 bp of the last exon plus the following 15 bp in the genome. If this 20 bp nucleotide window has more than 80% A's content, then we assume the presence of a genomic polyA.Repeat elements in the last exon and in the 5' and 3'end of thequery cDNA are identified. The repeats in these regions are searched as follow: those overlapping the last block/exon of a cDNA, those overlapping the first 50 bp in the 5'upstream region, and repeats overlapping the terminal 50 bp from the 3'downstream region. All information is stored for the later generation of composite features, rules and quality tests evaluation Figure CAFTAN compares the coordinates of mapped cDNAs after filtering with the genomic coordinates of reference sets from the publicly available resources of the Vertebrate Genome Annotation (VEGA) database  and ENSEexon score" defined as the ratio between the exons overlap and the sum of their lengths, is calculated for all transcript exons and query cDNA exons. Once the exons have been compared all to all a "transcript score" given below is calculated for every VEGA and/or ENSEMBL transcript found to overlap with the query cDNA.Each analyzed cDNA is linked to the best matching gene in VEGA and/or ENSEMBL Figure . Since VTranscript score = /(mrna_exon_nr + cdna_ex_nr));total_transcript_exon" score is the sum of all transcript exon scores and "mrna_exon_nr" and "cdna_ex_nr" are the number of exons in the transcript and in the query cDNA, respectively. The score can have values between 0 and 1, where 1 indicates a perfect match in all exons from the VEGA/ENSEMBL transcript to all the exons of the query cDNA and the same number of exons. The transcript with the highest score is selected as the best overlapping one to the query cDNA. All its relevant information is stored.Here the "The extracted features are combined to calculate further relevant information for each cDNA sequences Figure .First the mapping of a cDNA sequence to the genome is analyzed in detail. The percentage of a cDNA sequence that can be mapped to the genome gives an indication of possible colligation events during cDNA cloning, potential errors in the genome assembly, or the existence of trans-splicing. All information obtained from the raw BLAT output and its filtering is used to calculate the quality of the mapping process by taking into account the number of mismatched positions internally and in the 3' and 5' ends Table .The tests related to the cDNA structure determine if the query cDNA was the product of a single exon or a multi-exon gene and is calculated using the filtered BLAT Exon results.the number of repeats in the three regions described before in section 2.1.3, is analysed and post-processed to distinguish between simple repeats, low complexity repeats , and complex repeats . The repeat type composite feature in the corresponding region depends on how many repeats were found and how much they could influence the right cloning of the cDNA: Complex repeats > Simple repeats > Low complexity repeats.Once the splice type of each intron in a cDNA has been defined, the Ss type of the whole cDNA is then defined Table . The Ss-The number and type of signals are calculated for each cDNA using PolyAsignal and the results are used to create the feature signal type Table . The sigA polyA contamination is true if there is a polyA stretch in the genome at the 3' mapping position of the terminal cDNA exon, this is classified as a genomic polyA. Then the 3' end of the last exon is checked for the presence of complex and simple repeats within its last 50 bp or 20 bp respectively. If repeats are found in these regions the cDNA has "repeat contamination". If more than one repeat is found, only one will be shown with the following preference: First complex repeats in the following order, Alu > L1 > SVA > LTR, then simple repeats and last low complexity repeats."Mixed contamination" means both types, ie. genomic polyA and repeat contamination. The type of any putative contamination is classified and stored Table .The distribution of simple and composite features extracted from the training set  was studied depending on their tag- in the curated DKFZ set-  using clustering methods. We found distinct relationships among features that were able to characterize different sets of observations in the training set. These profiles -relationships among features- were discussed and some times modified according to the curator's expert knowledge. The decision rules obtained are presented in Table 1.) "Uncovered", i.e. those cDNAs that mapped with less than 20 % of their length to the genome, which usually means the presence of too many gaps in the genome 2.) "Bad_se", i.e. bad single exon cDNAs; 3.) "Bad_me", i.e. bad multiple exon cDNAs; 4.) "Questionable_se", and 5.) "Questionable_me", are cDNAs with single or multiple exons, respectively, that should be manually inspected by the curator/scientist in order to take a decision about their quality; 6.) "Good_se_cdna", i.e., high quality single exon cDNAs; 7.) "Good_me_cdna", i.e., high quality multiple exon cDNAs; 8) "Partial_mapped", are cDNAs with gaps bigger than 20 bp either in the 3', 5' end or in the internal region 9.) "Undefined", are any cDNAs that do not fit into one of the previous categories because they failed to pass the evaluation pipeline. Elements in the latter group will be used for the further refinement of the evaluation and classification system.The quality assessment starts with the evaluation of the quality of a cDNA mapping to the genome. The decision process is similar for multiple and single exon cDNAs Table . HoweverA cDNA is rated to be of high quality when it is perfectly mapped to the genome, has a polyA signal and a polyA tail, and in the case of multiple exons the Ss score is higher than 60 % and the Ss type is "Good splice sites" Table . The Ss cDNAs with one type of contamination repeats, or genomic polyA and/or lacking the polyA tail need to fulfill other criteria in order not to be classified as "Bad" Table . The preThe lack of a polyA tail at the 3' end is also negatively considered during the quality assessment process, because a polyA tail is a canonical motif that strongly promotes translation initiation through secondary structures, such as hairpins . And morbest_transcript score" > 0.98) to the query cDNA, then the cDNA will be classified as "Good" or "Questionable". If the match is not perfect ("best_transcript score" <= 0.98) the classification will depend on the presence/absence/type of contamination and/or the presence or absence of polyA signal and polyA tail  – around 70% – are found to be near CpG islands .We generated and analyzed two cDNA sets in order to evaluate the performance of our method. The first set contained 5,954 "known protein-coding" cDNAs extracted from the human VEGA database, which is the current "golden" annotation standard in mammalians. For comparison we created a set of 3,000 "bad" cDNAs sequences. To do this, we selected from the human genome i) randomly a chromosome, ii) the beginning of the cDNA according to the length of the randomly selected chromosome, and iii) a random number of exons, introns and their lengths. Limits for the introns and exons lengths were taken from the work of Sakharkar et al., . RandomlCAFTAN was written using Object Oriented Perl, and has been implemented as a downloadable stand-alone version available at . PreprocCAFTAN is also available under the W3H task system at the DKFZ . This WeThe Polyasignal source code is available through the authors and it has been submitted to be included in future releases of the EMBOSS package. Queries in the ENSEMBL database can be done "remote" at the ENSEMBL site or "locally" provided the user has installed the ENSEMBL databases. The default is set to remote.CAFTAN allows the analysis of several thousands of cDNAs within a few minutes, giving the curator/scientist an overview about the likelihood of having fully spliced/processed cDNAs and providing the already existing annotation for the cDNA set. It supports the rejection of low quality cDNAs and helps selecting novel splice variants, and/or completely novel transcripts for new experiments.The evaluation described in the implementation section to assess the performance of CAFTAN showed that it is able to correctly classify more than 85% of 5950 selected "known protein-coding" VEGA cDNAs as high quality multi- or single-exon Table . It idenWhen we analyzed the VEGA cDNAs classified by CAFTAN as "Bad_se" (37/191) and "Bad_multiple_exons" 791/5759) we found, that in the case of the group classified as "bad_multiple_exon_cdna", all of them lacked the presence of a polyA signal but had a perfect match to VEGA transcripts. The manual inspection by an expert curator of these VEGA CDNAs sequences showed that most of the VEGA cDNAs sequences were not complete, part of the 3'UTR region failing or having an artefact 3'UTR end. In Table 759 we fo"Splice Sites Score" is the composite features that provided the most reliable information about the quality of multi-exon cDNAs.It is worth mentioning that the Arabidopsis thaliana by Hayden et, al[When analyzing the remaining "bad" multiple exons  cDNAs, we saw that most of the wrong classification was due to mistakes in the mapping process. BLAT is the current fastest algorithm to map sequences to a genome. It is designed to find sequences of 95% and greater similarity in a minimum of 33 bases, thus it may miss shorter sequence alignments, making the mapping of shorter exons especially problematic. An alternative would be the use of MEGABLAST , and theen et, al. This apen et, al. Other pen et, al or like en et, al are alsoen et, al. This biSeveral cDNAs were wrongly classified because of misalignments in the first and last exons that were due to the presence of repeat elements in the UTR regions, and to misalignments of 3' terminal poly-A sequences. All these cDNAs were classified as being of questionable quality. Any cDNAs in this category should not be discarded but a rating in this category should rather be an indicator to the scientist curator that he or she should look in detail in the CAFTAN output and to take an expert decision on the quality of the query cDNA. The cDNAs in this category were about 5% in the multi-exon class of cDNAs.The transcript score, which evaluates the total overlap between a query cDNA and the VEGA/ENSEMBL annotated transcripts, was for both cDNAs types -when using the human NCBI assembly 35- between 1 and 0.8 in 99.5 % of the cases, where 1 is a perfect match. The results presented here, are based on assembly 36 and so when looking in the VEGA annotation, the average of the transcript score is lower. This is due to the fact that VEGA still uses the assembly NCBI35, while ENSEMBL and all the databases available for BLAT use the assembly NCBI36. This is a temporal problem that affects only to those chromosomes, which changed the mapping coordinates in the NCBI36 and will be solved in the moment that VEGA changes to the assembly NCBI36. In the random generated cDNAs sets all transcript scores remained under 0.23 %.CAFTAN was implemented as a fast approach towards the selection of cDNAs from resources that have been generated in high-throughput projects. A more accurate algorithm like MegaBlast would certainly improve the alignments, but this would be at the cost of speed. In view of the high success rate of CAFTAN in the analysis of the VEGA and random cDNA sets already obtained with BLAT, we decided not to compromise speed for a possible small improvement in reliability. Nevertheless, future implementations of CAFTAN will be allowing users to upload either BLAT or MegaBlast outputs or they can already use cDNA2Genome, which annotates cDNAs using this approach but which needs long running times. Both the WEB and the downloadable versions are modular and will further permit the implementation of additional filtering tools for alternative mapping methods.Mapping" output contains the exact mapping information of the cDNAs to the selected genome after filtering of the BLAT exons. The "Evaluation" output classifies the cDNAs according to their quality and the presence or lack of the features described above. It provides the following fields: contamination, number of mismatches in the 5' and 3'end, mapping classification, exon number, splice site number, score and type, polyA signal number type and score, repeats, repeats in the 3' end. "VEGA annotation" and "ENSEMBL annotation" outputs contain detailed annotation of genes and transcripts in VEGA/ENSEMBL overlapping the analyzed cDNAs. This file includes a quality score for each overlapping gene and transcript that is based on the number of shared positions over the total length of the overlapping annotated gene/transcript. The table also contains gene identifiers, begin and end of mapping positions, chromosome number, number of exons, and information on shared and missed exons. "VEGA detailed annotation" and "ENSEMBL detailed annotation" outputs provide further details at the individual exon level, plus positional differences between annotated VEGA/ENSEMBL exons and exons of the query cDNA. The "Polyasignal" output contains the results of the search for polyA signals and polyA tails in the cDNAs using the selected regular expressions. The final output of CAFTAN is an "XML" file that contains all information from all outputs obtained in the process. This XML file can be directly integrated in user databases and used for further analysis with other programs.Additionally to the cDNA quality classification, CAFTAN provides extensive information about each cDNA, which is divided in different tab tabulated outputs tables Figure . The "MaCurrently we are working on the development of supervised learning algorithms for the improvement of cDNA quality prediction, especially for single exon cDNAs. In the future CAFTAN will be additionally extended towards the classification of transcripts that are products of exon skipping events, alternative usage of Poly (A) sites, and the presence of alternative promoters and transcription start sites (TSS).The results described here were obtained with the default parameters established for CAFTAN and optimized for mammals. One of the advantages of CAFTAN is that it allows the adjustment of parameters to adapt it to any organism. The parameters can easily be changed in the configuration file (Config.pm). The polyAsignal program can be configured for other genome preferences, and the rules to be taken into account can be modified for different genomes. New organism's genomic databases can be used as soon as they become available by adding them to the configuration file. We think that it is a very useful program for the annotation and research of unfinished genomes. The use of CAFTAN as a first filtering step should be followed by an ORF analysis like described by Takeda et al. . We furtWe have developed a new high-throughput sequence analysis tool, CAFTAN see , which p- Existing databases in ENSEMBL- The need of a BLAT mapping output.- The performance of CAFTAN depends on the number of input cDNAs and on the overall machine load. Analysis of the 4,000 cDNAs from the VEGA set took 21.89 seconds on a six processor SUN Enterprise. The remote use of the MSQL ENSEMBL database could be slowed down when the ENSEMBL server is extensively accessed, favoring a local implementation.Operating system(s): Linux, UnixProgramming language: Perl5Requirements:- For the download distribution it is necessary to install ENSEMBL package, bioperl-2.1 or higher, and the Perl DBI Package prior to the installation of CAFTAN.- Use of the web version requires a HUSAR account at .License:Restrictions to use by non-academics: license neededV.K conceived the initial idea. C.V, V.K and KH.G developed and designed the project. C.V and P.E designed and coded the program Polyasignal, and designed CAFTAN classes as well as the downloadable module. C.V. developed the tests, coded the program, created the test sets, and drafted the manuscript. A. H-W created the XML schema for CAFTAN. A.P, S.W and S.S participated in coordination; curated manually the "bad" cDNAs set and helped to draft the manuscript. All authors read and approved the final version of the manuscript.Table of DKFZ annotated sequencesClick here for fileHand annotation of the DKFZ cDNAs sequencesClick here for filepolyasignal executable for EMBOSSClick here for filepolyasignal acd file for EMBOSSClick here for fileCPAN distribution of the CAFTAN softwareClick here for file