BACKGROUND
seven years after the first drafts of the human genome were published  <cit>  and three years after the completion of sequencing the human genome, the exact number of the protein-coding genes encoded in this genome is still unknown: the most likely estimates range between  <dig> – <dig>  genes  <cit> .
more significantly, recent analyses have shown that the exact genomic structure of human protein-coding genes is correctly predicted for only about 50– <dig> % of the genes  <cit> .
in other words, despite significant advances in computational gene identification, correct prediction of the genomic structure of the protein-coding genes of higher eukaryotes is still a very difficult task.
the main objective of our mispred project is to develop tools that can be used to identify mispredicted genes/proteins, primarily from metazoan genomes, in order to inform scientists of the reliability of predictions and to improve the quality of predictions.
the key question is: are there signs that may indicate that the predicted structure of a protein-coding gene might be erroneous?
the mispred approach is based on the principle that a protein-coding gene is likely to be mispredicted if some of its features  conflict with our current knowledge about protein-coding genes and proteins.
as a proof of principle, in the present work we describe five approaches – based on five dogmas – to identify suspicious proteins that are likely to be abnormal or mispredicted.
accordingly, the current version of mispred contains five routines, each focusing on a special type of conflict with one of the dogmas.
conflict with the dogma that the subcellular localization of extracellular and transmembrane proteins is defined by the presence of appropriate sequence signals.
the validity of this dogma is supported by studies on various disease-causing mutations, indicating that the absence of functional signal peptides prevents the translocation of proteins across the endoplasmic reticulum membrane and the mislocalized protein is rapidly degraded by the proteasome  <cit> .
similarly, the loss of functional transmembrane helices is known to lead to the mislocalization of membrane proteins  <cit> .
a major reason for the rapid degradation of mislocalized extracellular proteins is that their extracellular  domains are misfolded in the reductive milieu of the cytoplasm and are recognized and degraded by the protein quality control system of the cell  <cit> .
in the current version of mispred we used domain families the members of which occur only in the extracellular space  to identify proteins that are completely or partially extracellular.
the justification for this approach is that certain  domain families are known to have adapted  to the extracellular space: they occur exclusively in extracellular proteins or extracytoplasmic parts of transmembrane proteins  <cit> .
following identification of extracellular or transmembrane proteins containing such extracellular 'marker' domains, we examined whether they have the sequence signals  that could target these domains to the extracellular space.
according to this dogma, proteins that contain obligatory extracellular domains but lack appropriate sequence signals  are considered suspicious  since their obligatory extracellular domains are not delivered to the extracellular space where they are stable and properly folded.
conflict with the dogma that transmembrane proteins containing both cytoplasmic and extracellular parts have at least one transmembrane segment that passes through the cell membrane.
in the current version of mispred, we used protein domain families the members of which occur exclusively in the extracellular space and exclusively in the cytoplasmic space to identify transmembrane proteins and we asked whether these proteins possess regions that pass through the cell membrane.
according to this dogma, proteins that contain both obligatory extracellular and obligatory cytoplasmic domains but lack transmembrane segment separating them are considered erroneous.
conflict with the dogma that obligatory extracellular and obligatory nuclear domains do not co-occur in a single, multidomain protein  <cit> .
according to this dogma, proteins that contain both obligatory extracellular and obligatory nuclear domains are considered abnormal and nonviable since they cannot be delivered to a cellular compartment where both types of domains would be correctly folded and fully functional.
conflict with the rule that the protein fold is highly conserved in a domain family, therefore the number of amino acid residues in closely related members of a globular domain family usually fall into a relatively narrow range  <cit> .
this phenomenon reflects the fact that the highly cooperative, rapid folding of protein domains is the result of natural selection  <cit> , therefore insertion/deletion of larger segments into/from protein domains may yield macromolecules that are unable to rapidly adopt a correctly folded, viable and stable three-dimensional structure.
accordingly, proteins containing domains that consist of a significantly larger or smaller number of residues than closely related members of the same family may be suspected to be abnormal and nonviable.
conflict with the dogma that a protein is encoded by exons located on a single chromosome.
according to this dogma, chimeric proteins whose parts are encoded by two or more different genes located on distinct chromosomes are considered abnormal.
in the present work we describe the results of mispred analyses of various public databases and discuss the values and limitations of the mispred approach.
RESULTS
validation of the mispred approach on the swiss-prot section of the uniprotkb
the swiss-prot section of uniprotkb is the gold standard of protein databases therefore we have used swiss-prot as the benchmark with which to validate the concepts behind the mispred approach.
in view of the high quality of this manually curated database our original expectation was that very few, if any, of the swiss-prot entries are truly erroneous therefore it would provide a useful dataset with which to test the specificity of the different mispred routines.
mispred analyses of human, mouse, rat, chick, zebrafish, worm and fly swiss-prot entries have indeed identified very few swiss-prot entries as truly erroneous .
the details of the analyses of the swiss-prot entries are described in additional file  <dig>  and the list of the erroneous entries is deposited in additional file  <dig> .
the majority of these errors could be corrected by targeted search of genomic and est databases; the protocol used for the correction of errors will be described in another publication .
*values for suspicious, false positive and true positive sequences are expressed as percentage of the proteins relevant for the given conflict.
the majority of truly erroneous sequences were returned for conflicts  <dig> and  <dig>  however, these accounted for only  <dig> – <dig> % and  <dig> – <dig> % of the sequences of the different species, respectively.
there were three major types of true positives among the swiss-prot entries identified by conflict 1:
1) fragments of full-length proteins that are not known to be fragments and/or are not annotated as such in the database.
for example, lplc4_human  proved to be a fragment and its missing signal peptide could be predicted with the help of the sequence of a full-length mouse ortholog .
similarly, the sequence of c209c_mouse   could be corrected by targeted search of mouse genomic and est sequences .
2) mispredicted proteins.
the hypothetical worm protein yl15_caeel  is an example for this type of error.
the protein arose through in silico fusion of a gene related to the homeobox protein hm07_caeel  and a gene related to the kunitz_bpti containing protein cbg <dig>  q619j1_caebr  .
3) proteins translated from aberrant transcripts that do not encode viable proteins.
for example noe2_mouse  lacks a signal peptide, whereas the rat ortholog  and a different isoform of this mouse protein  do possess a signal sequence.
the mispred routine used for the detection of conflict  <dig> is characterized by a very low number of false positives.
there are three main sources of false positives:
1) some proteins are identified as suspicious due to the limitations of the bioinformatic tools incorporated in this mispred routine .
2) exceptions to the dogma on which this mispred routine is based, i.e.
some secreted proteins truly lack secretory signal peptides since they are subject to leaderless protein secretion  <cit> , such as the secreted proteins gapr1_human  and tinag_human .
3) exceptions to the rule that all members of an extracellular domain family are restricted to the extracellular space.
mispred routine based on conflict  <dig> also identified a number of truly erroneous entries.
a major source for this type of error is that the swiss-prot entry corresponds to an incomplete protein .
for example, the sequence of epha5_rat  contains only a fragment of a sam_ <dig> domain since the protein sequence is truncated at the c-terminal end .
the error in epha5_rat could be corrected by targeted search of the rat genome using the sequences of the full-length orthologs .
the routine based on conflict  <dig> identified no erroneous mouse, chicken, worm or fruitfly swiss-prot proteins and the proportion of trans-chromosomal chimeras is very low in the case of human  and rat  sequences.
on the other hand, the frequency of suspected chimeras is relatively high in the case of zebrafish .
as discussed in additional file  <dig> , the most likely explanation for this observation is that they are false positives: the chromosomal assignment of contigs encoding different parts of these zebrafish genes may not be correct.
mispred analyses have revealed that none of the swiss-prot entries violate the rules underlying conflicts  <dig> and  <dig>
the fact that the number of swiss-prot entries identified by mispred as erroneous is very low attests to both the high quality of this database and the reliability of the mispred approach.
assuming that the vast majority of the swiss-prot sequences that were not detected by mispred routines are true negatives the false positive rate of the routines was calculated to be ≤  <dig> , i.e.
their specificity is very high .
mispred analysis of the trembl section of uniprotkb
the primary motivation for mispred analysis of uniprotkb/trembl was that trembl entries are used in various types of evidence-based, extrinsic gene prediction programs and thus have a strong influence on the quality of gene predictions.
the results of the analyses of human proteins are summarized in table  <dig>
*values for suspicious, false positive and true positive sequences are expressed as percentage of the proteins relevant for the given conflict.
nd – not determined
the data shown in table  <dig> indicate that the proportion of suspicious trembl entries is relatively high in the case of conflict  <dig>  conflict  <dig> and conflict  <dig>  importantly, these values are orders of magnitude higher than those for the swiss-prot entries, indicating that the vast majority of trembl proteins identified by mispred as suspicious are truly erroneous.
the majority  of human trembl proteins containing at least one extracellular domain were found by mispred to lack a signal peptide and/or a transmembrane segment in contrast to  <dig> % in the case of human swiss-prot entries.
similarly,  <dig> % of human trembl entries containing at least one member of the pfam-a domain families suitable for the study of domain integrity were found to contain a domain of abnormal size, while this value is only  <dig> % in swiss-prot.
the reason why a high proportion of trembl proteins are identified by conflict  <dig> as suspicious is that many trembl entries are truncated at the n-terminal end and n-terminally truncated secreted proteins are likely to lack the signal peptides.
similarly, the high proportion of trembl entries affected by conflict  <dig> reflects the severe contamination of this database with proteins predicted for incomplete cdnas.
since cdnas are more likely to be incomplete at their 5' end than their 3' end, the size of pfam-a domains at the n-terminal end of proteins of the trembl database was found to deviate more significantly from the average size than those of internal domains , again indicating that a relatively large proportion of trembl entries are truncated at the n-terminal end.
in harmony with this explanation, 95%  and 100%  of the suspicious entries are also annotated as fragments in trembl.
errors of trembl entries are not only due to the incompleteness of cdnas; transcripts formed through aberrant splicing and chimeric transcripts may also contribute to errors in this database.
interestingly, there are numerous human trembl entries that are chimeric , different segments of the predicted protein sequences being encoded by different genes located on different chromosomes.
a large proportion  of these chimeric entries are annotated as resulting from the fusion of genes located on different chromosomes through chromosomal translocation in a cancer cell line,  <dig> % have no such annotation although the corresponding cdnas were cloned from cancer tissues.
it should be pointed out, however, that there are many chimeric proteins in uniprotkb derived from cdnas that were cloned from apparently normal tissues , suggesting that chimera formation is more general than previously thought.
for example, the cdna of the hypothetical protein flj <dig> , cloned from colon mucosa, is a chimera of two genes located on chromosome  <dig> and chromosome  <dig>  the n-terminal part of the protein is derived from the gene encoding the pr domain zinc finger protein  <dig>  , the c-terminal part is derived from the gene encoding the liver form of fatty acid-binding protein   .
another factor that increased the number of human chimeric proteins in trembl is that the biotechnology industry has contributed numerous synthetic  human constructs to the trembl database .
it is also noteworthy that the rate of chimeras is much higher in the case of zebrafish sequences  than in the case of other vertebrates.
as discussed in additional file  <dig> , the most likely explanation for this observation is that the chromosomal assignment of contigs encoding these zebrafish genes may not be correct: contigs carrying different fragments of a zebrafish gene may be incorrectly assigned to different chromosomes.
mispred routines for conflict  <dig> and conflict  <dig> identified no trembl entries as erroneous.
this is primarily due to the fact that sequences predicted in silico  are absent from this section of uniprotkb.
mispred analysis of the ensembl database and the gnomon-predicted proteins of the ncbi database
*values for suspicious, false positive and true positive sequences are expressed as percentage of the proteins relevant for the given conflict.
nd – not determined
*values for suspicious, false positive and true positive sequences are expressed as percentage of the proteins relevant for the given conflict.
nd – not determined
as illustrated in tables  <dig> and  <dig>  a relatively high proportion of ensembl and gnomon-predicted entries are detected by the routine for conflict  <dig> as suspicious .
the most likely explanation for this is that in a large proportion of secreted vertebrate proteins the signal peptide is encoded by an exon separated by a long intron from the downstream exon  <cit> .
low sequence conservation of signal peptides and low transcript- and est-coverage of 5' parts of protein-coding genes explain why finding short exons encoding just the signal peptides has a rather low rate of success in the case of vertebrate genomes.
this problem is less serious in the case of intron-poor genomes, such as those of caenorhabditis elegans or drosophila melanogaster since their signal peptides are less likely to be encoded by short, distinct exons  <cit> ; this is reflected in a lower proportion of suspicious proteins in the case of ensembl entries of these species.
furthermore, worm and fruitfly were the first metazoan organisms whose genome sequences were determined  <cit>  and genome annotation efforts of nearly a decade have significantly improved the quality of gene predictions.
very few erroneous ensembl and gnomon-predicted sequences were detected by the routine for conflict  <dig>  the explanation for the low rate of this type of error is that – unlike signal peptide segments – transmembrane helices are usually encoded by longer exons that also encode other conserved parts of transmembrane proteins, facilitating their detection.
another factor that facilitates detection of exons of transmembrane regions is that they are more likely to be located in the middle or 3' parts of genes whose transcript- and est-coverage is relatively high.
the true positives for conflict  <dig> were found to be of two major types:
the predicted protein lacks transmembrane helices since the corresponding region of the gene is mispredicted.
for example, ensxetp <dig> , which corresponds to the frog ortholog of ephrin receptor a <dig>  lacks a typical transmembrane helix between its extracellular and cytoplasmic domains; the missing transmembrane sequence could be corrected using frog est sequences .
detailed analysis of this group of true positives  revealed that the regions containing their transmembrane helices are encoded by relatively short exons distinct from those encoding conserved extracellular and cytoplasmic domains  <cit> , making it difficult to find these exons.
the gene was mispredicted by in silico fusion of distinct, tandem genes encoding extracellular and cytoplasmic proteins.
several examples of this type of error were found among fugu rubripes proteins, but not in the case of other organisms, including zebrafish.
a possible explanation for this observation is that the intergenic distance is significantly shorter in the compact genome of pufferfish than in the case of other vertebrate genomes  <cit> , increasing the chance of in silico fusion of tandem genes.
mispred routine for conflict  <dig> detected very few errors in predicted proteins.
analyses of these sequences have revealed that they arose as a result of in silico fusion of two or more distinct, tandem genes encoding extracellular and nuclear proteins.
interestingly, proteins containing extracellular pentaxin and nuclear chromo domains were found among human, mouse, rat and chicken ensembl proteins.
there are several interpretations for their occurrence in different warm-blooded animals.
one possible explanation is that since the constituent genes are closely linked in all these species, gene-prediction erroneously fused these otherwise independent genes.
an alternative explanation is that these genes truly give rise to novel transcripts and proteins in which nuclear chromo domains are fused to extracellular pentaxin domains.
in other words, nuclear chromo domains can co-occur with the extracellular pentaxin domains, either because the chromo domain is not an obligatory nuclear domain or the pentaxin domain is not an obligatory extracellular domain.
it is noteworthy in this respect that chen and bixby  <cit>  have cloned three mouse variants of neuronal pentraxin with chromo domain   but none of them have signal peptides suggesting that, unlike the major products of the neuronal pentraxin genes, they are not secreted.
it is still possible, however, that the fusion proteins encoded by chimeric transcripts are abnormal in the sense that they are non-viable.
it is important to point out that such chimeric transcripts of neuronal pentraxin are very rare, or absent, as revealed by blast searches of est databases.
the fact that the rate of such errors is highest in the case of fugu rubripes may be partly due to the short intergenic distance in the compact pufferfish genome.
mispred routine for conflict  <dig> detected a large number of erroneous proteins both among ensembl entries  and among gnomon-predicted entries  containing members of pfam-a domain families suitable for the study of domain integrity.
the relatively high rate of erroneous insertion or omission of exons encoding parts of domains indicates that misprediction of exons encoding pfam-a domains is quite general.
no erroneous human ensembl protein was detected by the routine for conflict  <dig>  i.e.
none of them were chimeras of genes located on different chromosomes.
this is not surprising in view of the fact that chromosomal assembly of human genomic contigs is reliable therefore ensembl is exempt from the error of trans-chromosomal prediction of human genes.
mispred analysis of the homo sapiens gnomon-predicted entries has identified only one sequence, xp_ <dig> , as a chimera of genomic regions located on chromosomes  <dig> and  <dig>  note that in the ncbi database xp_ <dig> has been replaced recently by the nonchimeric np_ <dig> , encoded on chromosome  <dig>  in contrast with this, several zebrafish entries were identified as suspicious by the mispred routine for conflict  <dig>  an observation best explained by errors in the chromosomal assignment and assembly of zebrafish contigs.
for example, ensdarp <dig>  aligns over its entire length with mammalian casein kinase i isoform gamma- <dig> proteins, but it is encoded by contigs assigned to chromosomes  <dig> and  <dig>  xp_ <dig>  corresponds to a fragment of the zebrafish ortholog of ephrin receptor epha <dig>  it aligns over its entire length with these receptors, but it is encoded by contigs assigned to chromosome  <dig> and  <dig>  incorrect assembly of contigs may lead to the in silico fusion of regions located on different chromosomes.
for example, the major part of ensdarp <dig> , encoded on chromosome  <dig>  is homologous with mammalian solute carrier organic anion transporter family member 3a <dig> proteins, but an unrelated n-terminal extension of ensdarp <dig> is encoded on chromosome  <dig>  xp_ <dig> , a zebrafish protein annotated as similar to traf interacting protein is a chimera of traf-interacting protein and plasminogen related growth factor receptor  <dig>
in summary, gnomon-predicted sequences and ensembl sequences are quite similar inasmuch as similarly high proportion of suspicious proteins can be detected by conflict  <dig> and conflict  <dig> and fewer errors are detected by conflict  <dig>  conflict  <dig> and conflict  <dig> .
despite these similarities, there are some differences.
for example, in the case of conflict  <dig> several ensembl sequences were erroneous because the regions corresponding to their transmembrane helices were mispredicted, whereas this type of error was not found among the gnomon-predicted sequences.
in principle, such differences may reflect differences in the performance of the two gene prediction pipelines or differences in the gene populations covered by the two databases.
it should also be pointed out that ensembl is a comprehensive source of known genes and genes predicted with genewise  <cit> , as well as the corresponding transcripts and proteins whereas in the ncbi database transcripts/proteins predicted by gnomon are distinguished from those of known transcripts/proteins by unique  identifiers.
to permit a more direct comparison of the performance of the two gene prediction pipelines we have compared the results of mispred analyses only for those protein-coding genes for which both ensembl and gnomon have at least one prediction .
comparison of the two datasets confirmed that the two gene prediction pipelines are similar inasmuch as they suffer primarily from errors detectable by mispred routines for conflict  <dig> and conflict  <dig>  whereas the rates of errors detectable by routines for conflicts  <dig>   <dig> and  <dig> are very low.
nevertheless, there are minor differences between the ensembl and ncbi gene prediction pipelines: ensembl is more likely to fail in the identification of exons encoding transmembrane helices, whereas ncbi's gnomon appears to be more prone to fuse tandem genes in silico .
*values for suspicious, false positive and true positive sequences are expressed as percentage of the proteins relevant for the given conflict.
nd – not determined.
the data refer to human genes for which both gene prediction pipelines generated at least one gene model.
mispred analysis of gencode sequences
the encode  project aims to identify all functional elements in the human genome sequence.
the pilot phase of the project is focused on specified  <dig> megabases  of the human genome sequence  <cit> .
gencode is a sub-project of encode; its overall goal is to identify all protein-coding genes in the regions of the human genome selected within the encode project.
mispred analysis of the  <dig> gencode peptides with the routine for conflict  <dig> identified one peptide  as containing an extracellular  domain but lacking signal peptide and transmembrane segments.
blast searches revealed that ac <dig> - <dig> corresponds to the n-terminal part of cadh2_human  but, due to alternative splicing, it lacks the n-terminal secretory signal peptide.
it seems unlikely that this transcript encodes a viable protein since – in the absence of a secretory signal peptide – it may not be transported into the extracytoplasmic space.
mispred analysis of the gencode peptides for conflict  <dig> identified  <dig>  as containing an abnormally short or abnormally long pfam-a domain.
in the majority of cases the deviant domains are n-terminally or c-terminally truncated simply as a consequence of the incompleteness of the transcripts.
nevertheless, we identified several cases where the domain deviates from normal size as a result of alternative splicing  <cit> .
examples include ac <dig> - <dig>  , rp11-247a <dig> - <dig>  , xx-fw83563b <dig> - <dig>  , ap <dig> - <dig>  , rp11-298j <dig> - <dig>   and rp11-247a <dig> - <dig>   containing domains that deviate from normal size as a result of alternative splicing.
structural and functional analyses of the putative proteins encoded by these alternatively spliced transcripts suggest that, in many cases, the deviation from normal domain-size may not be compatible with the viability of these proteins, suggesting that the transcripts arose through aberrant splicing  <cit> .
mispred analysis of the gencode peptides for conflicts  <dig>   <dig> and  <dig> did not identify suspicious sequences.
CONCLUSIONS
mispred may identify proteins as suspicious for three different reasons.
mispred may identify normal, viable proteins as suspicious due to errors of genomic data and limitations of the bioinformatic tools incorporated in mispred
mispred analyses of predicted zebrafish sequences illustrate the point that a 'correct' protein sequence may be suspected to be a chimera encoded by two or more genes located on different chromosomes if the assembly of the contigs is incorrect.
errors in the bioinformatic identification of sequence features used to detect the various types of conflicts may raise unjustified doubts about the viability of some protein sequences.
for example, this type of error is encountered in the case of some secreted or transmembrane proteins  whose atypical signal peptides or transmembrane helices are not detected with high confidence by the signal peptide or transmembrane helix prediction programs incorporated into the mispred routines.
similarly, the hmmpfam program may on occasion identify marker domains erroneously, leading to the incorrect prediction of the subcellular localization of a protein domain.
analyses of the benchmark swiss-prot proteins, however, have revealed that the false positive rates of the mispred routines are lower than  <dig> %.
for details of the specificity analyses see materials and methods.
mispred may identify normal, viable proteins as suspicious due to limitations of the dogmas on which mispred routines are based
a survey of the results of mispred analyses has revealed that there are some exceptions to the dogmas on which the mispred approach is based.
for example, some secreted proteins may truly lack secretory signal peptides since they are subject to leaderless protein secretion  <cit> , some predominantly extracellular, cytoplasmic or nuclear pfam-a domain families are not always restricted to a single subcellular location and thus may be multilocale etc.
similarly, it cannot be excluded at present that transchromosomal chimeras can be formed and may have normal physiological functions  <cit> .
nevertheless, the fact that mispred analyses of protein sequences of the swiss-prot database identified very few such exceptions indicates that the dogmas of mispred are generally valid.
mispred may identify truly abnormal, nonviable proteins
mispred analyses of swiss-prot, trembl, ensembl, ncbi and gencode sequences identified numerous hypothetical protein sequences that are likely to be nonviable since they violate some of the basic dogmas about viable proteins.
for example, analysis of the trembl sequences revealed that incomplete or abnormal hypothetical proteins translated in silico from incomplete cdnas or aberrant transcripts are quite abundant in this database.
a recent study  <cit>  has also revealed that alternative splicing frequently generates transcripts that encode nonviable proteins due to violation of domain integrity, loss of signal peptides, etc.
interestingly, there are also numerous chimeric entries in the trembl database, a large proportion of which are derived from normal tissues.
there are several mechanisms for the creation of chimeric proteins.
unequal crossing over resulting in the fusion of parts of tandem genes and chromosomal translocation resulting in the fusion of genes located on different chromosomes are the two best documented mechanisms for the creation of chimeric genes and proteins.
recent studies have convincingly shown that transcription of tandem genes into a single rna sequence and translation of the chimeric mrna into a chimeric protein  <cit>  is also a major mechanism for the creation of chimeric proteins.
unneberg and claverie  <cit>  have recently suggested that chimeric proteins might also be formed through transchromosomal transcription, i.e.
if genes located on different chromosomes are expressed in the same "transcription factory" they may give rise to chimeric transcripts.
it should be pointed out that the relatively high proportion of incomplete and abnormal entries in trembl also has an impact on gene prediction.
since most gene prediction pipelines rely on extrinsic information  <cit>  such as those provided by trembl, some of the errors of trembl may be inherited by the databases of predicted genes.
it is noteworthy in this respect that the majority of errors of ensembl and gnomon-predicted sequences can be detected with the use of conflict  <dig> and conflict  <dig>  probably reflecting the fact the trembl sequences also suffer from the same types of errors.
the relatively high proportion of transchromosomal chimeric sequences among trembl entries, however, does not have a major impact on gene prediction, provided that contig assembly is unambiguous.
sequences not detected by mispred do not necessarily correspond to normal, viable proteins
although mispred identifies many suspicious sequences, it should be emphasized that the routines detect only a fraction of the truly erroneous sequences.
first, the mispred routines described in this manuscript exploit only five of the various dogmas about viable proteins.
second, only a fraction of proteins contains members of well-characterized pfam-a domain families, i.e.
families on which the routines for conflicts  <dig>   <dig>   <dig> and  <dig> rely.
third, the number of suspicious sequences identified by conflicts  <dig>   <dig> or  <dig> significantly underestimates the actual number of sequences that may be affected by these types of errors since we used only validated, obligatory extracellular, cytoplasmic and nuclear pfam-a domain families to predict the subcellular localization of proteins and did not include numerous extracellular, cytoplasmic or nuclear domain families the members of which may also occur in other subcellular compartments.
fourth, in the case of conflict  <dig> mispred uses a high cut-off value: a domain is judged to be nonviable only if its pfam-a domain deviates from normal size by at least 40% in length.
for details of the sensitivity analyses see materials and methods.
summary
recent studies have shown that a significant proportion of eukaryotic genes may be mispredicted at the transcript level  <cit> .
since the mispred routines described here are able to detect many of these errors and may aid the correction of these errors we suggest that the mispred approach may significantly improve the quality of protein sequence data based on gene-predictions.
in order to increase the sensitivity of the approach we are currently developing additional routines, based on the violation of other dogmas about proteins.
the mispred approach may also be used as a discovery tool since it can also serve to explore the limitations of the dogmas on which the various mispred routines are based.

 seven years after the first drafts of the human genome were published and three years after the completion of sequencing the human genome, the exact number of the protein-coding genes encoded in this genome is still unknown: the most likely estimates range between – genes. more significantly, recent analyses have shown that the exact genomic structure of human protein-coding genes is correctly predicted for only about 50– % of the genes. in other words, despite significant advances in computational gene identification, correct prediction of the genomic structure of the protein-coding genes of higher eukaryotes is still a very difficult task. the main objective of our mispred project is to develop tools that can be used to identify mispredicted genes/proteins, primarily from metazoan genomes, in order to inform scientists of the reliability of predictions and to improve the quality of predictions. the key question is: are there signs that may indicate that the predicted structure of a protein-coding gene might be erroneous? the mispred approach is based on the principle that a protein-coding gene is likely to be mispredicted if some of its features conflict with our current knowledge about protein-coding genes and proteins. as a proof of principle, in the present work we describe five approaches – based on five dogmas – to identify suspicious proteins that are likely to be abnormal or mispredicted. accordingly, the current version of mispred contains five routines, each focusing on a special type of conflict with one of the dogmas. conflict with the dogma that the subcellular localization of extracellular and transmembrane proteins is defined by the presence of appropriate sequence signals. the validity of this dogma is supported by studies on various disease-causing mutations, indicating that the absence of functional signal peptides prevents the translocation of proteins across the endoplasmic reticulum membrane and the mislocalized protein is rapidly degraded by the proteasome. similarly, the loss of functional transmembrane helices is known to lead to the mislocalization of membrane proteins. a major reason for the rapid degradation of mislocalized extracellular proteins is that their extracellular domains are misfolded in the reductive milieu of the cytoplasm and are recognized and degraded by the protein quality control system of the cell. in the current version of mispred we used domain families the members of which occur only in the extracellular space to identify proteins that are completely or partially extracellular. the justification for this approach is that certain domain families are known to have adapted to the extracellular space: they occur exclusively in extracellular proteins or extracytoplasmic parts of transmembrane proteins. following identification of extracellular or transmembrane proteins containing such extracellular'marker' domains, we examined whether they have the sequence signals that could target these domains to the extracellular space. according to this dogma, proteins that contain obligatory extracellular domains but lack appropriate sequence signals are considered suspicious since their obligatory extracellular domains are not delivered to the extracellular space where they are stable and properly folded. conflict with the dogma that transmembrane proteins containing both cytoplasmic and extracellular parts have at least one transmembrane segment that passes through the cell membrane. in the current version of mispred, we used protein domain families the members of which occur exclusively in the extracellular space and exclusively in the cytoplasmic space to identify transmembrane proteins and we asked whether these proteins possess regions that pass through the cell membrane. according to this dogma, proteins that contain both obligatory extracellular and obligatory cytoplasmic domains but lack transmembrane segment separating them are considered erroneous. conflict with the dogma that obligatory extracellular and obligatory nuclear domains do not co-occur in a single, multidomain protein. according to this dogma, proteins that contain both obligatory extracellular and obligatory nuclear domains are considered abnormal and nonviable since they cannot be delivered to a cellular compartment where both types of domains would be correctly folded and fully functional. conflict with the rule that the protein fold is highly conserved in a domain family, therefore the number of amino acid residues in closely related members of a globular domain family usually fall into a relatively narrow range. this phenomenon reflects the fact that the highly cooperative, rapid folding of protein domains is the result of natural selection, therefore insertion/deletion of larger segments into/from protein domains may yield macromolecules that are unable to rapidly adopt a correctly folded, viable and stable three-dimensional structure. accordingly, proteins containing domains that consist of a significantly larger or smaller number of residues than closely related members of the same family may be suspected to be abnormal and nonviable. conflict with the dogma that a protein is encoded by exons located on a single chromosome. according to this dogma, chimeric proteins whose parts are encoded by two or more different genes located on distinct chromosomes are considered abnormal. in the present work we describe the results of mispred analyses of various public databases and discuss the values and limitations of the mispred approach.  validation of the mispred approach on the swiss-prot section of the uniprotkb the swiss-prot section of uniprotkb is the gold standard of protein databases therefore we have used swiss-prot as the benchmark with which to validate the concepts behind the mispred approach. in view of the high quality of this manually curated database our original expectation was that very few, if any, of the swiss-prot entries are truly erroneous therefore it would provide a useful dataset with which to test the specificity of the different mispred routines. mispred analyses of human, mouse, rat, chick, zebrafish, worm and fly swiss-prot entries have indeed identified very few swiss-prot entries as truly erroneous. the details of the analyses of the swiss-prot entries are described in additional file and the list of the erroneous entries is deposited in additional file. the majority of these errors could be corrected by targeted search of genomic and est databases; the protocol used for the correction of errors will be described in another publication. *values for suspicious, false positive and true positive sequences are expressed as percentage of the proteins relevant for the given conflict. the majority of truly erroneous sequences were returned for conflicts and however, these accounted for only – % and – % of the sequences of the different species, respectively. there were three major types of true positives among the swiss-prot entries identified by conflict 1: 1) fragments of full-length proteins that are not known to be fragments and/or are not annotated as such in the database. for example, lplc4_human proved to be a fragment and its missing signal peptide could be predicted with the help of the sequence of a full-length mouse ortholog. similarly, the sequence of c209c_mouse could be corrected by targeted search of mouse genomic and est sequences. 2) mispredicted proteins. the hypothetical worm protein yl15_caeel is an example for this type of error. the protein arose through in silico fusion of a gene related to the homeobox protein hm07_caeel and a gene related to the kunitz_bpti containing protein cbg q619j1_caebr. 3) proteins translated from aberrant transcripts that do not encode viable proteins. for example noe2_mouse lacks a signal peptide, whereas the rat ortholog and a different isoform of this mouse protein do possess a signal sequence. the mispred routine used for the detection of conflict is characterized by a very low number of false positives. there are three main sources of false positives: 1) some proteins are identified as suspicious due to the limitations of the bioinformatic tools incorporated in this mispred routine. 2) exceptions to the dogma on which this mispred routine is based, i.e. some secreted proteins truly lack secretory signal peptides since they are subject to leaderless protein secretion, such as the secreted proteins gapr1_human and tinag_human. 3) exceptions to the rule that all members of an extracellular domain family are restricted to the extracellular space. mispred routine based on conflict also identified a number of truly erroneous entries. a major source for this type of error is that the swiss-prot entry corresponds to an incomplete protein. for example, the sequence of epha5_rat contains only a fragment of a sam_ domain since the protein sequence is truncated at the c-terminal end. the error in epha5_rat could be corrected by targeted search of the rat genome using the sequences of the full-length orthologs. the routine based on conflict identified no erroneous mouse, chicken, worm or fruitfly swiss-prot proteins and the proportion of trans-chromosomal chimeras is very low in the case of human and rat sequences. on the other hand, the frequency of suspected chimeras is relatively high in the case of zebrafish. as discussed in additional file, the most likely explanation for this observation is that they are false positives: the chromosomal assignment of contigs encoding different parts of these zebrafish genes may not be correct. mispred analyses have revealed that none of the swiss-prot entries violate the rules underlying conflicts and the fact that the number of swiss-prot entries identified by mispred as erroneous is very low attests to both the high quality of this database and the reliability of the mispred approach. assuming that the vast majority of the swiss-prot sequences that were not detected by mispred routines are true negatives the false positive rate of the routines was calculated to be ≤, i.e. their specificity is very high. mispred analysis of the trembl section of uniprotkb the primary motivation for mispred analysis of uniprotkb/trembl was that trembl entries are used in various types of evidence-based, extrinsic gene prediction programs and thus have a strong influence on the quality of gene predictions. the results of the analyses of human proteins are summarized in table *values for suspicious, false positive and true positive sequences are expressed as percentage of the proteins relevant for the given conflict. nd – not determined the data shown in table indicate that the proportion of suspicious trembl entries is relatively high in the case of conflict conflict and conflict importantly, these values are orders of magnitude higher than those for the swiss-prot entries, indicating that the vast majority of trembl proteins identified by mispred as suspicious are truly erroneous. the majority of human trembl proteins containing at least one extracellular domain were found by mispred to lack a signal peptide and/or a transmembrane segment in contrast to % in the case of human swiss-prot entries. similarly, % of human trembl entries containing at least one member of the pfam-a domain families suitable for the study of domain integrity were found to contain a domain of abnormal size, while this value is only % in swiss-prot. the reason why a high proportion of trembl proteins are identified by conflict as suspicious is that many trembl entries are truncated at the n-terminal end and n-terminally truncated secreted proteins are likely to lack the signal peptides. similarly, the high proportion of trembl entries affected by conflict reflects the severe contamination of this database with proteins predicted for incomplete cdnas. since cdnas are more likely to be incomplete at their 5' end than their 3' end, the size of pfam-a domains at the n-terminal end of proteins of the trembl database was found to deviate more significantly from the average size than those of internal domains, again indicating that a relatively large proportion of trembl entries are truncated at the n-terminal end. in harmony with this explanation, 95% and 100% of the suspicious entries are also annotated as fragments in trembl. errors of trembl entries are not only due to the incompleteness of cdnas; transcripts formed through aberrant splicing and chimeric transcripts may also contribute to errors in this database. interestingly, there are numerous human trembl entries that are chimeric, different segments of the predicted protein sequences being encoded by different genes located on different chromosomes. a large proportion of these chimeric entries are annotated as resulting from the fusion of genes located on different chromosomes through chromosomal translocation in a cancer cell line, % have no such annotation although the corresponding cdnas were cloned from cancer tissues. it should be pointed out, however, that there are many chimeric proteins in uniprotkb derived from cdnas that were cloned from apparently normal tissues, suggesting that chimera formation is more general than previously thought. for example, the cdna of the hypothetical protein flj, cloned from colon mucosa, is a chimera of two genes located on chromosome and chromosome the n-terminal part of the protein is derived from the gene encoding the pr domain zinc finger protein, the c-terminal part is derived from the gene encoding the liver form of fatty acid-binding protein. another factor that increased the number of human chimeric proteins in trembl is that the biotechnology industry has contributed numerous synthetic human constructs to the trembl database. it is also noteworthy that the rate of chimeras is much higher in the case of zebrafish sequences than in the case of other vertebrates. as discussed in additional file, the most likely explanation for this observation is that the chromosomal assignment of contigs encoding these zebrafish genes may not be correct: contigs carrying different fragments of a zebrafish gene may be incorrectly assigned to different chromosomes. mispred routines for conflict and conflict identified no trembl entries as erroneous. this is primarily due to the fact that sequences predicted in silico are absent from this section of uniprotkb. mispred analysis of the ensembl database and the gnomon-predicted proteins of the ncbi database *values for suspicious, false positive and true positive sequences are expressed as percentage of the proteins relevant for the given conflict. nd – not determined *values for suspicious, false positive and true positive sequences are expressed as percentage of the proteins relevant for the given conflict. nd – not determined as illustrated in tables and a relatively high proportion of ensembl and gnomon-predicted entries are detected by the routine for conflict as suspicious. the most likely explanation for this is that in a large proportion of secreted vertebrate proteins the signal peptide is encoded by an exon separated by a long intron from the downstream exon. low sequence conservation of signal peptides and low transcript- and est-coverage of 5' parts of protein-coding genes explain why finding short exons encoding just the signal peptides has a rather low rate of success in the case of vertebrate genomes. this problem is less serious in the case of intron-poor genomes, such as those of caenorhabditis elegans or drosophila melanogaster since their signal peptides are less likely to be encoded by short, distinct exons; this is reflected in a lower proportion of suspicious proteins in the case of ensembl entries of these species. furthermore, worm and fruitfly were the first metazoan organisms whose genome sequences were determined and genome annotation efforts of nearly a decade have significantly improved the quality of gene predictions. very few erroneous ensembl and gnomon-predicted sequences were detected by the routine for conflict the explanation for the low rate of this type of error is that – unlike signal peptide segments – transmembrane helices are usually encoded by longer exons that also encode other conserved parts of transmembrane proteins, facilitating their detection. another factor that facilitates detection of exons of transmembrane regions is that they are more likely to be located in the middle or 3' parts of genes whose transcript- and est-coverage is relatively high. the true positives for conflict were found to be of two major types: the predicted protein lacks transmembrane helices since the corresponding region of the gene is mispredicted. for example, ensxetp, which corresponds to the frog ortholog of ephrin receptor a lacks a typical transmembrane helix between its extracellular and cytoplasmic domains; the missing transmembrane sequence could be corrected using frog est sequences. detailed analysis of this group of true positives revealed that the regions containing their transmembrane helices are encoded by relatively short exons distinct from those encoding conserved extracellular and cytoplasmic domains, making it difficult to find these exons. the gene was mispredicted by in silico fusion of distinct, tandem genes encoding extracellular and cytoplasmic proteins. several examples of this type of error were found among fugu rubripes proteins, but not in the case of other organisms, including zebrafish. a possible explanation for this observation is that the intergenic distance is significantly shorter in the compact genome of pufferfish than in the case of other vertebrate genomes, increasing the chance of in silico fusion of tandem genes. mispred routine for conflict detected very few errors in predicted proteins. analyses of these sequences have revealed that they arose as a result of in silico fusion of two or more distinct, tandem genes encoding extracellular and nuclear proteins. interestingly, proteins containing extracellular pentaxin and nuclear chromo domains were found among human, mouse, rat and chicken ensembl proteins. there are several interpretations for their occurrence in different warm-blooded animals. one possible explanation is that since the constituent genes are closely linked in all these species, gene-prediction erroneously fused these otherwise independent genes. an alternative explanation is that these genes truly give rise to novel transcripts and proteins in which nuclear chromo domains are fused to extracellular pentaxin domains. in other words, nuclear chromo domains can co-occur with the extracellular pentaxin domains, either because the chromo domain is not an obligatory nuclear domain or the pentaxin domain is not an obligatory extracellular domain. it is noteworthy in this respect that chen and bixby have cloned three mouse variants of neuronal pentraxin with chromo domain but none of them have signal peptides suggesting that, unlike the major products of the neuronal pentraxin genes, they are not secreted. it is still possible, however, that the fusion proteins encoded by chimeric transcripts are abnormal in the sense that they are non-viable. it is important to point out that such chimeric transcripts of neuronal pentraxin are very rare, or absent, as revealed by blast searches of est databases. the fact that the rate of such errors is highest in the case of fugu rubripes may be partly due to the short intergenic distance in the compact pufferfish genome. mispred routine for conflict detected a large number of erroneous proteins both among ensembl entries and among gnomon-predicted entries containing members of pfam-a domain families suitable for the study of domain integrity. the relatively high rate of erroneous insertion or omission of exons encoding parts of domains indicates that misprediction of exons encoding pfam-a domains is quite general. no erroneous human ensembl protein was detected by the routine for conflict i.e. none of them were chimeras of genes located on different chromosomes. this is not surprising in view of the fact that chromosomal assembly of human genomic contigs is reliable therefore ensembl is exempt from the error of trans-chromosomal prediction of human genes. mispred analysis of the homo sapiens gnomon-predicted entries has identified only one sequence, xp_, as a chimera of genomic regions located on chromosomes and note that in the ncbi database xp_ has been replaced recently by the nonchimeric np_, encoded on chromosome in contrast with this, several zebrafish entries were identified as suspicious by the mispred routine for conflict an observation best explained by errors in the chromosomal assignment and assembly of zebrafish contigs. for example, ensdarp aligns over its entire length with mammalian casein kinase i isoform gamma- proteins, but it is encoded by contigs assigned to chromosomes and xp_ corresponds to a fragment of the zebrafish ortholog of ephrin receptor epha it aligns over its entire length with these receptors, but it is encoded by contigs assigned to chromosome and incorrect assembly of contigs may lead to the in silico fusion of regions located on different chromosomes. for example, the major part of ensdarp, encoded on chromosome is homologous with mammalian solute carrier organic anion transporter family member 3a proteins, but an unrelated n-terminal extension of ensdarp is encoded on chromosome xp_, a zebrafish protein annotated as similar to traf interacting protein is a chimera of traf-interacting protein and plasminogen related growth factor receptor in summary, gnomon-predicted sequences and ensembl sequences are quite similar inasmuch as similarly high proportion of suspicious proteins can be detected by conflict and conflict and fewer errors are detected by conflict conflict and conflict. despite these similarities, there are some differences. for example, in the case of conflict several ensembl sequences were erroneous because the regions corresponding to their transmembrane helices were mispredicted, whereas this type of error was not found among the gnomon-predicted sequences. in principle, such differences may reflect differences in the performance of the two gene prediction pipelines or differences in the gene populations covered by the two databases. it should also be pointed out that ensembl is a comprehensive source of known genes and genes predicted with genewise, as well as the corresponding transcripts and proteins whereas in the ncbi database transcripts/proteins predicted by gnomon are distinguished from those of known transcripts/proteins by unique identifiers. to permit a more direct comparison of the performance of the two gene prediction pipelines we have compared the results of mispred analyses only for those protein-coding genes for which both ensembl and gnomon have at least one prediction. comparison of the two datasets confirmed that the two gene prediction pipelines are similar inasmuch as they suffer primarily from errors detectable by mispred routines for conflict and conflict whereas the rates of errors detectable by routines for conflicts and are very low. nevertheless, there are minor differences between the ensembl and ncbi gene prediction pipelines: ensembl is more likely to fail in the identification of exons encoding transmembrane helices, whereas ncbi's gnomon appears to be more prone to fuse tandem genes in silico. *values for suspicious, false positive and true positive sequences are expressed as percentage of the proteins relevant for the given conflict. nd – not determined. the data refer to human genes for which both gene prediction pipelines generated at least one gene model. mispred analysis of gencode sequences the encode project aims to identify all functional elements in the human genome sequence. the pilot phase of the project is focused on specified megabases of the human genome sequence. gencode is a sub-project of encode; its overall goal is to identify all protein-coding genes in the regions of the human genome selected within the encode project. mispred analysis of the gencode peptides with the routine for conflict identified one peptide as containing an extracellular domain but lacking signal peptide and transmembrane segments. blast searches revealed that ac - corresponds to the n-terminal part of cadh2_human but, due to alternative splicing, it lacks the n-terminal secretory signal peptide. it seems unlikely that this transcript encodes a viable protein since – in the absence of a secretory signal peptide – it may not be transported into the extracytoplasmic space. mispred analysis of the gencode peptides for conflict identified as containing an abnormally short or abnormally long pfam-a domain. in the majority of cases the deviant domains are n-terminally or c-terminally truncated simply as a consequence of the incompleteness of the transcripts. nevertheless, we identified several cases where the domain deviates from normal size as a result of alternative splicing. examples include ac -, rp11-247a -, xx-fw83563b -, ap -, rp11-298j - and rp11-247a - containing domains that deviate from normal size as a result of alternative splicing. structural and functional analyses of the putative proteins encoded by these alternatively spliced transcripts suggest that, in many cases, the deviation from normal domain-size may not be compatible with the viability of these proteins, suggesting that the transcripts arose through aberrant splicing. mispred analysis of the gencode peptides for conflicts and did not identify suspicious sequences.  mispred may identify proteins as suspicious for three different reasons. mispred may identify normal, viable proteins as suspicious due to errors of genomic data and limitations of the bioinformatic tools incorporated in mispred mispred analyses of predicted zebrafish sequences illustrate the point that a'correct' protein sequence may be suspected to be a chimera encoded by two or more genes located on different chromosomes if the assembly of the contigs is incorrect. errors in the bioinformatic identification of sequence features used to detect the various types of conflicts may raise unjustified doubts about the viability of some protein sequences. for example, this type of error is encountered in the case of some secreted or transmembrane proteins whose atypical signal peptides or transmembrane helices are not detected with high confidence by the signal peptide or transmembrane helix prediction programs incorporated into the mispred routines. similarly, the hmmpfam program may on occasion identify marker domains erroneously, leading to the incorrect prediction of the subcellular localization of a protein domain. analyses of the benchmark swiss-prot proteins, however, have revealed that the false positive rates of the mispred routines are lower than %. for details of the specificity analyses see materials and methods. mispred may identify normal, viable proteins as suspicious due to limitations of the dogmas on which mispred routines are based a survey of the results of mispred analyses has revealed that there are some exceptions to the dogmas on which the mispred approach is based. for example, some secreted proteins may truly lack secretory signal peptides since they are subject to leaderless protein secretion, some predominantly extracellular, cytoplasmic or nuclear pfam-a domain families are not always restricted to a single subcellular location and thus may be multilocale etc. similarly, it cannot be excluded at present that transchromosomal chimeras can be formed and may have normal physiological functions. nevertheless, the fact that mispred analyses of protein sequences of the swiss-prot database identified very few such exceptions indicates that the dogmas of mispred are generally valid. mispred may identify truly abnormal, nonviable proteins mispred analyses of swiss-prot, trembl, ensembl, ncbi and gencode sequences identified numerous hypothetical protein sequences that are likely to be nonviable since they violate some of the basic dogmas about viable proteins. for example, analysis of the trembl sequences revealed that incomplete or abnormal hypothetical proteins translated in silico from incomplete cdnas or aberrant transcripts are quite abundant in this database. a recent study has also revealed that alternative splicing frequently generates transcripts that encode nonviable proteins due to violation of domain integrity, loss of signal peptides, etc. interestingly, there are also numerous chimeric entries in the trembl database, a large proportion of which are derived from normal tissues. there are several mechanisms for the creation of chimeric proteins. unequal crossing over resulting in the fusion of parts of tandem genes and chromosomal translocation resulting in the fusion of genes located on different chromosomes are the two best documented mechanisms for the creation of chimeric genes and proteins. recent studies have convincingly shown that transcription of tandem genes into a single rna sequence and translation of the chimeric mrna into a chimeric protein is also a major mechanism for the creation of chimeric proteins. unneberg and claverie have recently suggested that chimeric proteins might also be formed through transchromosomal transcription, i.e. if genes located on different chromosomes are expressed in the same "transcription factory" they may give rise to chimeric transcripts. it should be pointed out that the relatively high proportion of incomplete and abnormal entries in trembl also has an impact on gene prediction. since most gene prediction pipelines rely on extrinsic information such as those provided by trembl, some of the errors of trembl may be inherited by the databases of predicted genes. it is noteworthy in this respect that the majority of errors of ensembl and gnomon-predicted sequences can be detected with the use of conflict and conflict probably reflecting the fact the trembl sequences also suffer from the same types of errors. the relatively high proportion of transchromosomal chimeric sequences among trembl entries, however, does not have a major impact on gene prediction, provided that contig assembly is unambiguous. sequences not detected by mispred do not necessarily correspond to normal, viable proteins although mispred identifies many suspicious sequences, it should be emphasized that the routines detect only a fraction of the truly erroneous sequences. first, the mispred routines described in this manuscript exploit only five of the various dogmas about viable proteins. second, only a fraction of proteins contains members of well-characterized pfam-a domain families, i.e. families on which the routines for conflicts and rely. third, the number of suspicious sequences identified by conflicts or significantly underestimates the actual number of sequences that may be affected by these types of errors since we used only validated, obligatory extracellular, cytoplasmic and nuclear pfam-a domain families to predict the subcellular localization of proteins and did not include numerous extracellular, cytoplasmic or nuclear domain families the members of which may also occur in other subcellular compartments. fourth, in the case of conflict mispred uses a high cut-off value: a domain is judged to be nonviable only if its pfam-a domain deviates from normal size by at least 40% in length. for details of the sensitivity analyses see materials and methods. summary recent studies have shown that a significant proportion of eukaryotic genes may be mispredicted at the transcript level. since the mispred routines described here are able to detect many of these errors and may aid the correction of these errors we suggest that the mispred approach may significantly improve the quality of protein sequence data based on gene-predictions. in order to increase the sensitivity of the approach we are currently developing additional routines, based on the violation of other dogmas about proteins. the mispred approach may also be used as a discovery tool since it can also serve to explore the limitations of the dogmas on which the various mispred routines are based. 
analyses of predicted ensembl protein sequences of nine deuterostome and two protostome species have revealed that the absence of expected signal peptides and violation of domain integrity account for the majority of mispredictions. the current version of the mispred approach uses five distinct routines for identifying abnormal, incomplete or mispredicted entries based on the principle that a sequence is likely to be incorrect if some of its features conflict with our current knowledge about protein-coding genes and proteins: conflict between the predicted subcellular localization of proteins and the absence of the corresponding sequence signals; presence of extracellular and cytoplasmic domains and the absence of transmembrane segments; co-occurrence of extracellular and nuclear domains; violation of domain integrity; chimeras encoded by two or more genes located on different chromosomes. we suggest that application of the mispred approach will significantly improve the quality of gene predictions and the associated databases.
 despite significant improvements in computational annotation of genomes, sequences of abnormal, incomplete or incorrectly predicted genes and proteins remain abundant in public databases. since the majority of incomplete, abnormal or mispredicted entries are not annotated as such, these errors seriously affect the reliability of these databases. here we describe the mispred approach that may provide an efficient means for the quality control of databases. the current version of the mispred approach uses five distinct routines for identifying abnormal, incomplete or mispredicted entries based on the principle that a sequence is likely to be incorrect if some of its features conflict with our current knowledge about protein-coding genes and proteins: conflict between the predicted subcellular localization of proteins and the absence of the corresponding sequence signals; presence of extracellular and cytoplasmic domains and the absence of transmembrane segments; co-occurrence of extracellular and nuclear domains; violation of domain integrity; chimeras encoded by two or more genes located on different chromosomes.  analyses of predicted ensembl protein sequences of nine deuterostome and two protostome species have revealed that the absence of expected signal peptides and violation of domain integrity account for the majority of mispredictions. analyses of sequences predicted by ncbi's gnomon annotation pipeline show that the rates of mispredictions are comparable to those of ensembl. interestingly, even the manually curated uniprotkb/swiss-prot dataset is contaminated with mispredicted or abnormal proteins, although to a much lesser extent than uniprotkb/trembl or the ensembl or gnomon-predicted entries.  mispred works efficiently in identifying errors in predictions generated by the most reliable gene prediction tools such as the ensembl and ncbi's gnomon pipelines and also guides the correction of errors. we suggest that application of the mispred approach will significantly improve the quality of gene predictions and the associated databases. 