BACKGROUND
gene expression microarray technology plays an important role in modern biomedical research.
as a result of technological innovations it is possible to measure gene expression of ten thousands of gene products at the same time.
consequently, studies using these techniques generate huge amounts of data which need to be properly analyzed.
for a correct biological interpretation, knowledge of what the reporters on the array actually measure is very important.
it is quite common that a substantial fraction of the used reporters are annotated with less useful descriptions like "expressed sequence tag"  or "hypothetical gene".
in the worst case no useful information about the reporter sequences is provided at all.
to overcome this problem, annotation procedures should be directed towards finding the most informative description.
microarray reporter sequences are either oligonucleotide reporter sequences designed using nucleotide databases such as embl  <cit> /genbank  <cit> , unigene  <cit>  andlkdjkfjd, ensembl  <cit>  or refseq  <cit> , or clone sequences  that need to be sequenced before they can be annotated.
microarray developers usually provide the sequences from which the oligonucleotide sequences were derived, or the sequences on the array itself, alternatively they give the database accession number of the first blast-hit  <cit>  of either of these sequences.
to improve the biological understanding of large-scale gene expression studies it is helpful to determine whether the products of the regulated genes have known biological functions or structures, or are known to be involved in metabolic or regulatory pathways.
the common approach to achieve this is to visualize these genes as part of gene groups or pathways.
information needed for this kind of gene classification can be found in the gene ontology database  <cit> , or derived from literature or protein databases.
pathways used by scientists are found for instance in kegg   <cit> , in genmapp  mapp archives  <cit>  and at biocarta  <cit> , but such pathways are not always ideally suited for a thorough interpretation of the microarray data.
for instance, kegg pathways were constructed based on enzyme commission  numbers  <cit> .
it is not trivial to relate these ecs to functional proteins.
but, there are cases where one single ec actually groups multiple enzyme subtypes together.
for example, the human superoxide dismutase-enzyme  represents one single enzyme function in the enzyme database.
however, there are three different enzymes known with this function that are present in different parts of the cell .
these three sods do not only have different cofactor binding properties but are also known to have different structures and functions.
when a microarray analysis is performed at the pathway level, you do not want to associate all three enzymes with the same ec to a reaction that is actually performed by just one of them.
newer initiatives, such as reactome, start with identifying biomolecular reactions, group them together and construct molecular pathways around them  <cit> .
each reactome reaction description contains information about the proteins involved, usually through a reference to uniprot .
since both signaling and metabolic pathways are understood at the level of proteins involved, it is best to annotate both the pathway node in the pathway diagram and the array reporters with the id for the corresponding protein product.
a good candidate protein database to base this annotation upon is uniprot, providing curated, high quality information about the proteins and many references towards other interesting databases, such as omim  <cit> , entrez gene   <cit> , pdb  <cit>  and many more.
the most obvious approach towards finding a protein product for a microarray reporter sequence is by aligning the translated reporter sequence against a protein database by using the blastx-program  <cit> .
this approach is used in recently published annotation tools such as targetidentifier  <cit>  and garban  <cit> , that are aimed to identify protein products by improving the annotation of ests.
although the results of such an approach are not high in number, the quality of the hits is usually good.
however, there are two main reasons why this approach is not always successful:  mrna and gene sequences often contain large non-coding regions that cannot be translated into functional protein domains and therefore will not yield good high quality alignments.
the nucleotide sequences of clones often contain sequencing errors, leading to insertions or deletions.
this will lead to frame shifts in the translated protein sequence, resulting in two or more separate  hits for parts of the sequence when using blastx.
of course the common way to circumvent this translation problem is to blast the reporter sequence against a nucleotide database.
this approach is generally applied by commercial array providers  <cit> .
it yields a maximum amount of results, but the quality of the annotation found is not always optimal: alignments can point out to something similar to the actual sequence.
finding relatively non-informative sequences, such as ests, and annotation results where the gene is described as "being similar to something else" is also quite common.
this leads to two main problems: the quality of the hit and the quality of the annotation of the hit.
based on the above it is clear that a good annotation method must: a) find meaningful descriptions wherever possible b) not find descriptions where the actual alignment of reporter and annotated sequence  are not good enough c) find the best description available at the given time ; d) make use of an identifier that is commonly used in visualization and analytical tools.
the method we describe uses a newly derived database that was created by cleaning up a redundant embl  database with the help of crosslinks within uniprot.
these results were combined with a more traditional annotation method, based on blastn searches against ensembl, followed by a conversion towards their corresponding uniprot id.
in a last step, both methods were combined to obtain the final annotation table .
RESULTS
annotation results incyte mouse unigene i array
the incyte mouse unigene i array consists of  <dig>  clones of variable length.
for annotation purposes, incyte opted for unigene cluster annotation for most of the clones present as shown in figure 2a.
after converting the older unigene clusters to their uniprot counterpart a large number of clusters were found to be no longer present in the mouse subset of unigene release build  <dig>  however, for the unigene clusters that were still present in the current unigene database, it was possible to annotate more than half of them with uniprot ids.
when protein similarities with rat and/or human were also taken into account, this number increased slightly.
from the total number of reporter clone sequences present  <dig> % of the reporters could be coupled to a valid mouse uniprot id using the novel double crosslink approach, this number increased to  <dig> % when references to other species were included.
after combining both annotation approaches the total annotation rate ended up at  <dig> %.
these results are summarized in figure 2b and 2c.
a considerable number of reporters were originally annotated with less-informative descriptions  such as ests and riken cdna clones .
with the new approach we were able to associate  <dig>   of these reporters with one or more proteins.
when the ensembl references were included as well, this percentage increased to  <dig> %.
in total,  <dig>  reporters did not pass our high-quality criteria after being blasted against the primary species database, while  <dig> reporters aligned with more than one uniprot id
this table summarizes all categories of the originally less-informative descriptions on the incyte mouse unigene i array after reannotation.
we were able to annotate  <dig>   of those with a uniprot identifier.
in total  <dig>   clones were given a more specific description.
sp, swissprot/uniprot; ens, ensembl
for comparison we also annotated this array with two existing procedures.
when we used the blastx based targetidentifier approach on all  <dig>  incyte array clone sequences we were able to couple  <dig>  reporters with a uniprot id, of which  <dig>  mouse uniprot ids.
alternatively, a direct blastn against refseq was used which did find about 10% less annotations than our approach did, but was far less effective on the pathway level .
annotation results agilent g2519a option  <dig> mouse development 44k array
the agilent g2519a option  <dig> mouse development array contains  <dig>  60-mer reporters that were annotated with one or two national institute of aging  or national institute of health  mouse genome ids  <cit> .
a closer inspection revealed that less than half of the total reporter descriptions  contained meaningful gene information .
all other reporters  had a poor annotation, further illustrated in table  <dig>  the cembl approach identified  <dig> % of all  <dig>  features with a uniprot mouse id.
adding the results of the cembl blast against rat and human, this number increased to  <dig> % and  <dig> % respectively .
combining both ensembl and cembl annotation resulted properly identifying over  <dig> % of all features .
in total,  <dig> % of these features were linked to one or more mouse uniprot ids.
adding the protein information of both rat and human species increased this number to  <dig> % and  <dig> % respectively.
this table categorizes all originally less-informative feature descriptions on the agilent g2519a option  <dig> mouse development array  into several groups.
after blasting their corresponding sequences against either cembl or ensembl, we were able to relate  <dig>   features to an improved description.
for the "unknown" category more than half of the features now have an improved annotation.
of those, more than half refer to known proteins.
sp, swissprot/uniprot; ens, ensembl
most reporters on this array were originally associated with less-informative descriptions , as shown in table  <dig>  our annotation methods were able to functionally annotate  <dig> % of these reporters with one or more proteins.
this number increased to  <dig> % after adding the ensembl based annotation.
our methods coupled more than one uniprot id to  <dig> individual reporters.
for this array we also compared the results with a direct blastn against refseq and again our approach found about 10% more annotations.
comparing old versus new reporter annotations
for most of the array reporters both original and newly found descriptions were identical, but some reporters were annotated with either several functionally different proteins or with different protein family-members.
in some cases the new annotation updated the given description.
for example, "ras related protein rab" and "neuropilin and tolloid like" became respectively "ras-related protein rab-6b" and "neuropilin- <dig> precursor ".
the outcome of this comparison is for both arrays illustrated in figures 4a and 4b.
comparing our cembl versus refseq/targetidentifier annotations
to compare the results of each method  with our cembl approach,  <dig>  reporters were randomly selected for each array using the c++ random number generator.
when both methods yielded a protein hit, the reporter annotations were compared based on a match in either the gene name or in the protein description .
for over 90% of the array reporters that yielded a protein product using both the cembl and the refseq approach the same protein was found.
for some of the conflicting annotations our approach yielded more than one uniprot id and the corresponding one could be found lower in the list .
another part of these reporters linked to a different family member , whereas a small part of these array reporters did not overlap at all .
one conflicting reporter on the agilent array  was flagged as really bad since our approach found more than ten protein annotations and they did not correspond to the refseq protein.
for targetidentifier the overlap was somewhat smaller .
this was to be expected since targetidentifier aligns the sequences against more species than only our species of interest.
in general, our cembl method was able to find more meaningful hits  compared to the other approaches.
this is reflected by the number of reporters that were accepted in the genmapp gene database .
gene ontology and pathway visualization
this table summarizes for each approach all reporters that could be imported and visualized using the genmapp program.
a clear distinction has been made between the local mapps released on september 20th,  <dig> and the gene ontology database, built in the mappfinder program of genmapp .
in general, improving the annotation yields better pathway visualization.
for the incyte array , the genmapp  <dig>  program  <cit>  was able to link  <dig>  newly annotated reporters to unique go nodes, compared to  <dig>  reporters based on the original unigene annotation.
linking these reporters with  <dig> local mouse pathways included in the genmapp program, it was able to associate  <dig>  old unigene ids with unique genes present in their local database.
out of this amount, only  <dig> genes could be shown in a local pathway.
these numbers increased after applying our annotation methods to respectively  <dig>  and  <dig> , whereas the refseq approach ended up with  <dig>  proteins accepted in the gene database of which only  <dig>  could be found on a local pathway.
for the original agilent array annotation  it was possible to extract and convert  <dig>  reporters towards a usable refseq protein  identifier.
after applying our methods, there was not only a higher number of unique genes accepted in the genmapp database, but the amount of reporters visualized on go level increased from  <dig>  to  <dig>  as well.
shifting our focus towards the local pathway level, our methods added  <dig> extra reporters that now can be linked to a gene present in a local pathway.
the refseq approach found only about 10% fewer gene annotations  when compared to our approach.
converting these dna ids  to their protein counterparts yielded  <dig>  protein  annotations.
only  <dig>  of these refseq protein ids were accepted in the genmapp mouse gene database, visualizing  <dig>  genes on a local pathway and providing  <dig>  gene ontology classifications.
improving pathway visualization
a) classification of the incyte array reporters
these tables display the total number of individual reporters that were either accepted or rejected in the mouse gene database by importing both the original , the refseq  and our combined  annotation.
comparing the original annotation with our combined annotation approach;  comparing the refseq-based annotation with our combined annotation approach;
for the incyte array we successfully increased the reporters linked to unique genes in the gene database from  <dig>  to  <dig> .
surprisingly,  <dig> incyte reporters could originally be visualized using the old unigene annotation, but after applying our combined approaches they did not appear on any mapp.
of those  <dig> reporters:   <dig> reporters remained un-annotated.
re-evaluating the blast results of these reporters showed that about half of them  showed some alignment with a gene sequence , but these alignments failed our quality criteria.
<dig> reporters were associated with either a rat or human uniprot id and  <dig> with a rat or human ensembl id that could not be converted to uniprot.
because the genmapp mapps and gene database are species-specific, it was not possible to visualize the human and rat based annotations.
the remaining  <dig> reporters were linked to a mouse identifier that was not present in the mouse gene database.
b) classification of the agilent reporters
similarly for the agilent array the amount of reporters linked to unique genes in the genmapp gene database increased from  <dig>  to  <dig> .
after applying our new annotation methods, about  <dig>  probes could not be linked to pathway nodes again.
for these  <dig>  probes: a)  <dig>  probes remained un-annotated.
of those only  <dig> showed an alignment of > 70% with a gene sequence while the others only gave very poor hits.
b)  <dig> probes referenced in our new annotation to a rat or ruman identifier.
c)  <dig> probes were linked to mouse uniprot ids that were not available in the genmapp mouse gene database.
c) local pathway improvement evaluation using genmapp
the improvement of the percentages of genes that can be visualized in individual pathways is shown in figure  <dig>  using our annotation methods, the number of gene products that can be visualized increased on  <dig>  and  <dig>  out of  <dig> pathways compared to what could be achieved with the refseq-based annotation .
in general, our methods largely increased the amount of genes visualized on a number of important pathways.
to illustrate this improvement, one pathway is shown in figure  <dig> where the newly mapped gene products are shown in red, while the gene products that could be visualized using both the refseq and our new annotations are colored green.
the blue boxes represent genes that were only found using the direct refseq blastn approach.
discussion
when performing microarray experiments, where the expression of tens of thousands individual genes is measured simultaneously, it is important to correctly understand the biological outcome.
to achieve this, array reporters need to be annotated with the gene or gene product that they correspond with.
nowadays, array producers provide annotation files containing necessary information for their arrays, such as the database identifier of the gene of which the reporter was based on, the functional name of the gene  and/or the actual reporter sequence.
the quality of these annotation files can differ between array producers.
but even if the annotation quality is satisfactory at the moment of purchase, it is important to keep the annotation up to date.
gene expression data repositories like geo   <cit>  and arrayexpress  <cit>  contain more and more experimental data.
an important usage option for these repositories is to do future integrated analysis, combining different kinds of data.
before this can be done, it is crucial to have adequate and up-to-date annotations.
if these annotations are not updated while genome sequence databases are, we do not only lose part of the benefit of the improvements of genome annotations, but we will also lose information since older annotations do not necessarily couple to the data used in newer analytical tools.
this problem often occurs when the reporters are based on unigene ids.
for the incyte array used in this paper originally  <dig> % of the reporters were annotated with a unigene id.
a large fraction  of this annotation has become useless for pathway mapping by now since the cluster ids do not longer exist in the database.
this implies that it is important that databases keep track of older ids that have been used like, for instance, ensembl and unigene do, although unigene does not archive its obsolete cluster sequences.
nowadays, many annotation tools are available that aim to functionally annotate a given reporter id with a specific gene ontology  classification  <cit> .
a complementary approach is to interpret the data in a biological way by making use of pathway visualization tools, such as genmapp  <cit> , metacore  <cit>  and/or arrayxpath ii  <cit> .
these tools link an element in a pathway with a specific identifier, usually by an ensembl, uniprot, unigene, entrez gene or go id.
hence, to allow the biological interpretation in a pathway context, the goal should be to derive descriptions that contain the physiological most useful information for each reporter, i.e.
an identifier of a protein or other gene product.
some annotation methods use a blastx approach to find protein descriptions  <cit> .
other annotation tools do not include a blast search in their annotation procedure, but basically try to link entries from different databases to each other using a reference id  <cit> .
a few examples of these tools are the database for annotation, visualization and integrated discovery   <cit>  and the annotation builder library  present in the bioconductor module of r  <cit> , where the user has to supply a list of specific gene identifiers which will then be further linked towards other databases.
if the gene identifier involved is correctly coupled to the array reporter, then such tools will allow us to find database reference ids in numerous genomic databases.
if the gene identifier is not correctly coupled, then the biological interpretation will go astray  <cit> .
thus, for microarray experiments it is important to start from the beginning, i.e.
by blasting the reporter sequence used on the array.
during the last years much effort has been put in redefining the annotation of the affymetrix genechip arrays  <cit> .
a more recent paper by harbig et al.
describes how they re-blasted all affymetrix human genome u133a  <dig>  target sequences and compared them with the annotation provided by affymetrix.
their findings concluded that even for this often used chip about one third of the probe sets could be updated  <cit> .
furthermore, to facilitate biological understanding it is imperative to continually keep the annotation up-to-date and as accurate as possible, even if that means annotating a single reporter with more than one gene.
affymetrix is aware of this problem and use an "x_at"-tag at the end of the probeset name to indicate that that probeset can detect more than one gene.
but these x_at probe sets are always described with only one gene product and the other products are thus not normally included in the analysis.
a related problem exists when pathway visualization tools are used.
these tools do not offer a solution yet on how to visualize reporters that will hybridize strongly with more than one gene target.
when researchers do know all genes that could be detected by a specific reporter, they could validate these results by pcr-based techniques using all this information.
our method therefore aims to offer the end-user as much information as possible about reporter specificity: for reporters that detect more than one gene or protein product, the description, gene name and identifiers are given for all possible near-perfect alignments.
it is then up to the end-user to decide if they want to include the reporter in their further analysis procedure.
because of previous experiments performed on both the incyte unigene mouse i and the agilent g2519a option  <dig> mouse development 44k arrays we wanted to reanalyze the data and use existing pathway tools to see if more biological information could be extracted.
the existing array annotations for both arrays were problematic:  the incyte array contained many annotations relating to unigene clusters that have been removed from the unigene database and are therefore no longer useful;  the agilent annotation was based on the nia mouse cdna project  <cit> .
the  <dig>  reporters present on this array were derived from genomic mouse ests and clones, explaining the non-informative description for a large amount of the reporters present .
because existing annotation methods using the blastn algorithm  <cit>  did not fulfill our criteria, we decided to develop a new method that followed a few strict criteria.
uniprot was chosen as reference database because its entries are crosslinked to other databases that give relevant information for further analysis.
filtering the nucleotide embl database, by only holding on to references from within a species-specific uniprot subset, resulted in a less redundant and more meaningful cleaned embl  subset.
a direct blastn against this cembl yields hits that are directly related to protein information.
additionally, we performed a blastn against the transcripts present in the ensembl database and crosslinked the ensembl id back to end up with a uniprot id.
the ensembl project plays an important role in current bioinformatics related to microarray annotation.
it contains much information from genomic sequence up to the protein level.
identification of the relation between a microarray reporter and an ensembl gene also allows evaluation of other aspects described or referenced in ensembl like gene splicing, variants, snps and gene orthologs.
nowadays, there are many tools available that accept ensembl ids.
thus, if an ensembl id contains no crosslink to uniprot, that id can still be used for continued analysis.
this powerful feature makes ensembl an ideal candidate database to blast against.
traditionally annotation tools often used unigene as reference database  <cit> .
this is in principle not wrong, but we strongly discourage this approach since it is known that after each unigene update clusters are split, joined together or removed from the database, often resulting in obsolete annotation for that reporter gene.
additionally, the extra information coming from within unigene is more limited in situations where the complete genome of an organism has already been sequenced.
unigene based approaches, as described above for the older incyte annotations, are still useful for reporters that were historically annotated with unigene clusters and for species where ensembl builds are not yet available.
many labs still have clone collections for which they only have unigene annotations and no sequence information.
after applying our annotation methods the number of reporters annotated with a uniprot or an ensembl id on either array was over 60%.
this is quite high, considering the large number of reporters that originally had less-informative descriptions .
whenever the original annotation also lead to a meaningful description the content of the original and our new annotation was most often identical, although our methods updated the description of  <dig> %  and  <dig> %  of these reporters for instance to a specific protein family member.
for the incyte array a lot of unigene cluster ids did no longer exist, preventing further analysis relative to go terms and pathway mapping, even if the descriptions of the lost clusters was in principle correct.
this loss of unigene clusters can be explained by both an improvement in the clustering algorithms used and an increase in the amounts of sequence data processed.
furthermore, depositors can withdraw poor sequence data at a later time, resulting in unresolved sequences.
the old incyte annotation did give annotations for a number of reporters  that our new methods failed to annotate.
this is primarily due to the higher alignment criteria that we applied.
if these alignment criteria would be lowered, then the amount of reporters annotated would increase .
for agilent, the number of probes that our methods failed to reannotate amounted to  <dig> % of the total.
for about one third of these lost agilent annotations the original description was non-informative .
the rest originally gave specific for gene products, but could not be confirmed by our annotation methods.
the cembl database contributed a considerable amount of direct protein annotations for both arrays.
since thus derived annotations are more trustworthy than the ones built on the ensembl approach we preferably kept them whenever the two approaches yielded different uniprot results.
we compared the results of our approach with existing approaches.
first we looked at targetidentifier  <cit> .
this web service attempts to identify ests as full-length cdnas and to provide further functional annotation.
first, the est sequences are divided into categories, based on the presence and location of a start- and stop-codon and passed through a blastx algorithm.
this approach circumvents part of frame shift problems in a general blastx approach that were mentioned in the introduction as the position of start- and stop codon allows for a choice of a fixed frame.
where our approach found  <dig>  annotations for the incyte array, targetidentifier found only  <dig>  uniprot id's and only  <dig>  of these were from the correct species.
we also used a direct blast against refseq with the standard  e-value of  <dig>  and the same filtering for at least 90% alignment that we used in all other blast procedures.
the refseq approach improved the original annotation of the incyte array, but was less successful on the agilent mouse development 44k array.
our methods found about 10% extra annotation on both arrays compared to the refseq approach .
a much larger difference occurred when we looked for usability of this annotation in pathway mapping and gene ontology classification .
this is caused by the fact that genmapp uses an ensembl based gene database.
refseq protein ids  are only accepted if they are linked to ensembl genes in the ensembl database itself.
ensembl only includes the link between nucleotide and protein identifiers when this identity is confirmed.
for most  of the protein identifiers linked to refseq nm ids that are not found in ensembl this is not the case .
the remaining 10% is probably caused by the refseq and ensembl builds being out of sync .
to verify that the ensembl gene database is the cause of the lower acceptance of refseq ids we used biomart on build  <dig> of the mus musculus ensembl database to associate the refseq dna ids, resulting from blasting against refseq, with np  id's.
for both the agilent and incyte reporters the number of refseq np id's found in this way corresponded exactly with the number of genes accepted in the genmapp gene database starting from the larger numbers of np identifiers found in refseq through conversion in refseq itself.
.
the background of this problem is that we understand biological pathways as protein functionality, and the proteins that are part of a pathway are primarily described by this protein functionality  while array reporters are normally annotated with nucleotide products.
the mapping of proteins to corresponding genes in databases like refseq and ensembl is still generally better than the mapping of nucleotide sequences to proteins.
we tried to evaluate whether the increase in coverage comes from a decrease in specificity, meaning we annotated reporters that should not be annotated, or that we coupled the original nucleotide annotation to a protein where that is not correct.
using our alignment quality criteria the actual thermodynamic hybridization conditions correspond to a one basepair mismatch in a  <dig> base sequence.
this essentially is what is used for the affymetrix match/mismatch method and is known to lower hybrizations to a large extend .
as a result, false positives  are less likely than they are in approaches where the first blast hit is used with only an e-value cut-off.
database crosslinks from nucleotide databases to protein databases are often created automatically.
the crosslinks from protein databases  are highly curated and errors are less likely.
since the initial step in our procedure uses the crosslinks from uniprot to embl it makes sense that fewer false positives will occur.
to evaluate this further we compared the outcome of the annotation for  <dig>  reporters for our approach and the refseq and targetidentifier approaches in more detail .
in general, when protein descriptions for the alternative annotations are compared they are identical.
the most important differences are: 1) compared to the targetidentifier approach both our method and the refseq method found many more results.
this is due to the intrinsic limitations of the targetidentifier approach which is based on blastx.
also the targetidentifier approach sometimes finds hits for the wrong species which cannot be used in pathway analysis.
2) our approach was able to find protein descriptions where the refseq approach found only corresponding nucleotide sequences for 12–14% of the reporters.
3) the other way around, the refseq approach found 8–10% proteins that we only found as nucleotides.
most of these could in fact be mapped to the genmapp gene database as well using the nucleotide descriptions.
4) we found 5–10% extra protein descriptions where the refseq approach found nothing.
this is the most suspect group for possible false positives.
for this group we checked individual reporter descriptions for the hits that we found in cembl database.
these were shown to indeed contain a small number  of possible false positives as they pointed to shorter  bac clones.
these clones were included in the cembl database since they are referred from uniprot, but the actual hit might occur away from the relevant gene sequence.
while comparing the old and the new annotations we encountered some situations where a microarray reporter sequence corresponded with more than one protein: for example, agilent reporters a_66_p <dig> and a_66_p <dig> resulted in finding high quality alignment against several genes.
after filtering, both reporters yielded different unique uniprot ids.
for a_66_p <dig> they were respectively "histone h2a type 1" , "histone h2b type 1-b "  and three other family members, while for a_66_p <dig> they were "ankyrin repeat domain-containing protein 40"  and "cisplatin resistance-associated overexpressed protein" .
in both cases the conclusion would be that the specificity of the reporters' sequence is low, allowing it to detect either more than one family member or even two functionally very different proteins.
in the latter case, the reporter should be omitted from further analysis.
in some specific situation both ensembl and cembl found more than  <dig> different uniprot ids for  <dig> agilent reporters .
furthermore, the lists of proteins found showed no overlap for these reporters.
direct sequence analysis and evaluation of the annotations indicated that these reporter sequences were originally unknown or derived from riken cdna.
for these  <dig> agilent probes, it is clear their annotation should be omitted from further analysis.
this demonstrates that, next to finding the best possible description, reblasting the sequence adds an extra quality check for the reporter sequence used.
after the reporter sequences are annotated, pathway visualization tools can be used to increase understanding of the biological outcome such as the genmapp program.
the original unigene id's of the incyte array could be used directly for genmapp analysis, but this off course yielded poor results since many of those unigene id's are outdated.
for the agilent array we verified whether the old annotations could still provide useful results by the use of methods that would couple them to ontology terms: a) by using fatigo  <cit> , since the documentation explicitly claims that that webservice accepts agilent feature ids.
this approach was unsuccessful, because no single reporter could be linked to a go term.
b) by using the ensembl database, that contains crosslinks towards agilent reporters.
in the end, this approach was not feasible, since the probe associations stored in ensembl belong to a different array type .
we concluded that these methods were unable to answer our question, so a work-around was created that visualized these genes in genmapp  by converting the refseq dna identifiers directly to their refseq protein counterparts.
for only ~ <dig>  of the ~ <dig>  agilent reporters that contained a refseq reference in their original annotation were we able to find a refseq protein .
this was not very different from the numbers we got when we did a direct blastn against refseq for comparison.
using mappfinder, the results of our combined annotation approaches increased the amount of visualized gene products in both the local pathways and in the gene ontology classification.
for both arrays, there were a few exceptions where the refseq approach was able to visualize some genes which our approach could not.
the refseq approach found  <dig>  annotations that we did not find while we found  <dig>  reporters in the gene database that the refseq approach did not find .
a manual reblast of these sequences  against both databases indicated that the difference is mainly due to differences between the cdna sequences present in refseq and ensembl: i.e.
perfect hits against refseq versus no hit at all in ensembl.
apart from this, the improvement in the amount of gene products mapped was large: the number of genes that could be mapped to a local pathway increased for incyte from  <dig>  to  <dig>   to  <dig>  .
for the agilent array a blastn against refseq found fewer sequences than the original agilent annotation, probably because of the extra alignment criteria applied.
as a result it was possible to visualize a slightly smaller amount  of genes in the local pathways, compared to the original , and substantially less  than found with our approach.
even with these relatively small changes in numbers of annotated genes the visualization power increased for a quarter of all pathways when comparing the refseq approach to the original annotation, but decreased in about half of all pathways.
this increase was even stronger for the larger pathways.
a full list of these individual pathway rankings are available as additional file  <dig> or can be downloaded from our servers  <cit> .
CONCLUSIONS
the usage of a high quality check for each individual reporter annotation, by requesting > 90% alignment identify, ensures that with the method described only those gene products are included in the annotation that will really hybridize.
in general, the methods described in this paper increased the quality of the reporter annotations and increased the amount of annotated reporter sequences that can be visualized using pathway visualization tools.

 gene expression microarray technology plays an important role in modern biomedical research. as a result of technological innovations it is possible to measure gene expression of ten thousands of gene products at the same time. consequently, studies using these techniques generate huge amounts of data which need to be properly analyzed. for a correct biological interpretation, knowledge of what the reporters on the array actually measure is very important. it is quite common that a substantial fraction of the used reporters are annotated with less useful descriptions like "expressed sequence tag" or "hypothetical gene". in the worst case no useful information about the reporter sequences is provided at all. to overcome this problem, annotation procedures should be directed towards finding the most informative description. microarray reporter sequences are either oligonucleotide reporter sequences designed using nucleotide databases such as embl /genbank, unigene andlkdjkfjd, ensembl or refseq, or clone sequences that need to be sequenced before they can be annotated. microarray developers usually provide the sequences from which the oligonucleotide sequences were derived, or the sequences on the array itself, alternatively they give the database accession number of the first blast-hit of either of these sequences. to improve the biological understanding of large-scale gene expression studies it is helpful to determine whether the products of the regulated genes have known biological functions or structures, or are known to be involved in metabolic or regulatory pathways. the common approach to achieve this is to visualize these genes as part of gene groups or pathways. information needed for this kind of gene classification can be found in the gene ontology database, or derived from literature or protein databases. pathways used by scientists are found for instance in kegg, in genmapp mapp archives and at biocarta, but such pathways are not always ideally suited for a thorough interpretation of the microarray data. for instance, kegg pathways were constructed based on enzyme commission numbers. it is not trivial to relate these ecs to functional proteins. but, there are cases where one single ec actually groups multiple enzyme subtypes together. for example, the human superoxide dismutase-enzyme represents one single enzyme function in the enzyme database. however, there are three different enzymes known with this function that are present in different parts of the cell. these three sods do not only have different cofactor binding properties but are also known to have different structures and functions. when a microarray analysis is performed at the pathway level, you do not want to associate all three enzymes with the same ec to a reaction that is actually performed by just one of them. newer initiatives, such as reactome, start with identifying biomolecular reactions, group them together and construct molecular pathways around them. each reactome reaction description contains information about the proteins involved, usually through a reference to uniprot. since both signaling and metabolic pathways are understood at the level of proteins involved, it is best to annotate both the pathway node in the pathway diagram and the array reporters with the id for the corresponding protein product. a good candidate protein database to base this annotation upon is uniprot, providing curated, high quality information about the proteins and many references towards other interesting databases, such as omim, entrez gene, pdb and many more. the most obvious approach towards finding a protein product for a microarray reporter sequence is by aligning the translated reporter sequence against a protein database by using the blastx-program. this approach is used in recently published annotation tools such as targetidentifier and garban, that are aimed to identify protein products by improving the annotation of ests. although the results of such an approach are not high in number, the quality of the hits is usually good. however, there are two main reasons why this approach is not always successful: mrna and gene sequences often contain large non-coding regions that cannot be translated into functional protein domains and therefore will not yield good high quality alignments. the nucleotide sequences of clones often contain sequencing errors, leading to insertions or deletions. this will lead to frame shifts in the translated protein sequence, resulting in two or more separate hits for parts of the sequence when using blastx. of course the common way to circumvent this translation problem is to blast the reporter sequence against a nucleotide database. this approach is generally applied by commercial array providers. it yields a maximum amount of results, but the quality of the annotation found is not always optimal: alignments can point out to something similar to the actual sequence. finding relatively non-informative sequences, such as ests, and annotation results where the gene is described as "being similar to something else" is also quite common. this leads to two main problems: the quality of the hit and the quality of the annotation of the hit. based on the above it is clear that a good annotation method must: a) find meaningful descriptions wherever possible b) not find descriptions where the actual alignment of reporter and annotated sequence are not good enough c) find the best description available at the given time; d) make use of an identifier that is commonly used in visualization and analytical tools. the method we describe uses a newly derived database that was created by cleaning up a redundant embl database with the help of crosslinks within uniprot. these results were combined with a more traditional annotation method, based on blastn searches against ensembl, followed by a conversion towards their corresponding uniprot id. in a last step, both methods were combined to obtain the final annotation table.  annotation results incyte mouse unigene i array the incyte mouse unigene i array consists of clones of variable length. for annotation purposes, incyte opted for unigene cluster annotation for most of the clones present as shown in figure 2a. after converting the older unigene clusters to their uniprot counterpart a large number of clusters were found to be no longer present in the mouse subset of unigene release build however, for the unigene clusters that were still present in the current unigene database, it was possible to annotate more than half of them with uniprot ids. when protein similarities with rat and/or human were also taken into account, this number increased slightly. from the total number of reporter clone sequences present % of the reporters could be coupled to a valid mouse uniprot id using the novel double crosslink approach, this number increased to % when references to other species were included. after combining both annotation approaches the total annotation rate ended up at %. these results are summarized in figure 2b and 2c. a considerable number of reporters were originally annotated with less-informative descriptions such as ests and riken cdna clones. with the new approach we were able to associate of these reporters with one or more proteins. when the ensembl references were included as well, this percentage increased to %. in total, reporters did not pass our high-quality criteria after being blasted against the primary species database, while reporters aligned with more than one uniprot id this table summarizes all categories of the originally less-informative descriptions on the incyte mouse unigene i array after reannotation. we were able to annotate of those with a uniprot identifier. in total clones were given a more specific description. sp, swissprot/uniprot; ens, ensembl for comparison we also annotated this array with two existing procedures. when we used the blastx based targetidentifier approach on all incyte array clone sequences we were able to couple reporters with a uniprot id, of which mouse uniprot ids. alternatively, a direct blastn against refseq was used which did find about 10% less annotations than our approach did, but was far less effective on the pathway level. annotation results agilent g2519a option mouse development 44k array the agilent g2519a option mouse development array contains 60-mer reporters that were annotated with one or two national institute of aging or national institute of health mouse genome ids. a closer inspection revealed that less than half of the total reporter descriptions contained meaningful gene information. all other reporters had a poor annotation, further illustrated in table the cembl approach identified % of all features with a uniprot mouse id. adding the results of the cembl blast against rat and human, this number increased to % and % respectively. combining both ensembl and cembl annotation resulted properly identifying over % of all features. in total, % of these features were linked to one or more mouse uniprot ids. adding the protein information of both rat and human species increased this number to % and % respectively. this table categorizes all originally less-informative feature descriptions on the agilent g2519a option mouse development array into several groups. after blasting their corresponding sequences against either cembl or ensembl, we were able to relate features to an improved description. for the "unknown" category more than half of the features now have an improved annotation. of those, more than half refer to known proteins. sp, swissprot/uniprot; ens, ensembl most reporters on this array were originally associated with less-informative descriptions, as shown in table our annotation methods were able to functionally annotate % of these reporters with one or more proteins. this number increased to % after adding the ensembl based annotation. our methods coupled more than one uniprot id to individual reporters. for this array we also compared the results with a direct blastn against refseq and again our approach found about 10% more annotations. comparing old versus new reporter annotations for most of the array reporters both original and newly found descriptions were identical, but some reporters were annotated with either several functionally different proteins or with different protein family-members. in some cases the new annotation updated the given description. for example, "ras related protein rab" and "neuropilin and tolloid like" became respectively "ras-related protein rab-6b" and "neuropilin- precursor ". the outcome of this comparison is for both arrays illustrated in figures 4a and 4b. comparing our cembl versus refseq/targetidentifier annotations to compare the results of each method with our cembl approach, reporters were randomly selected for each array using the c++ random number generator. when both methods yielded a protein hit, the reporter annotations were compared based on a match in either the gene name or in the protein description. for over 90% of the array reporters that yielded a protein product using both the cembl and the refseq approach the same protein was found. for some of the conflicting annotations our approach yielded more than one uniprot id and the corresponding one could be found lower in the list. another part of these reporters linked to a different family member, whereas a small part of these array reporters did not overlap at all. one conflicting reporter on the agilent array was flagged as really bad since our approach found more than ten protein annotations and they did not correspond to the refseq protein. for targetidentifier the overlap was somewhat smaller. this was to be expected since targetidentifier aligns the sequences against more species than only our species of interest. in general, our cembl method was able to find more meaningful hits compared to the other approaches. this is reflected by the number of reporters that were accepted in the genmapp gene database. gene ontology and pathway visualization this table summarizes for each approach all reporters that could be imported and visualized using the genmapp program. a clear distinction has been made between the local mapps released on september 20th, and the gene ontology database, built in the mappfinder program of genmapp. in general, improving the annotation yields better pathway visualization. for the incyte array, the genmapp program was able to link newly annotated reporters to unique go nodes, compared to reporters based on the original unigene annotation. linking these reporters with local mouse pathways included in the genmapp program, it was able to associate old unigene ids with unique genes present in their local database. out of this amount, only genes could be shown in a local pathway. these numbers increased after applying our annotation methods to respectively and, whereas the refseq approach ended up with proteins accepted in the gene database of which only could be found on a local pathway. for the original agilent array annotation it was possible to extract and convert reporters towards a usable refseq protein identifier. after applying our methods, there was not only a higher number of unique genes accepted in the genmapp database, but the amount of reporters visualized on go level increased from to as well. shifting our focus towards the local pathway level, our methods added extra reporters that now can be linked to a gene present in a local pathway. the refseq approach found only about 10% fewer gene annotations when compared to our approach. converting these dna ids to their protein counterparts yielded protein annotations. only of these refseq protein ids were accepted in the genmapp mouse gene database, visualizing genes on a local pathway and providing gene ontology classifications. improving pathway visualization a) classification of the incyte array reporters these tables display the total number of individual reporters that were either accepted or rejected in the mouse gene database by importing both the original, the refseq and our combined annotation. comparing the original annotation with our combined annotation approach; comparing the refseq-based annotation with our combined annotation approach; for the incyte array we successfully increased the reporters linked to unique genes in the gene database from to. surprisingly, incyte reporters could originally be visualized using the old unigene annotation, but after applying our combined approaches they did not appear on any mapp. of those reporters: reporters remained un-annotated. re-evaluating the blast results of these reporters showed that about half of them showed some alignment with a gene sequence, but these alignments failed our quality criteria. reporters were associated with either a rat or human uniprot id and with a rat or human ensembl id that could not be converted to uniprot. because the genmapp mapps and gene database are species-specific, it was not possible to visualize the human and rat based annotations. the remaining reporters were linked to a mouse identifier that was not present in the mouse gene database. b) classification of the agilent reporters similarly for the agilent array the amount of reporters linked to unique genes in the genmapp gene database increased from to. after applying our new annotation methods, about probes could not be linked to pathway nodes again. for these probes: a) probes remained un-annotated. of those only showed an alignment of > 70% with a gene sequence while the others only gave very poor hits. b) probes referenced in our new annotation to a rat or ruman identifier. c) probes were linked to mouse uniprot ids that were not available in the genmapp mouse gene database. c) local pathway improvement evaluation using genmapp the improvement of the percentages of genes that can be visualized in individual pathways is shown in figure using our annotation methods, the number of gene products that can be visualized increased on and out of pathways compared to what could be achieved with the refseq-based annotation. in general, our methods largely increased the amount of genes visualized on a number of important pathways. to illustrate this improvement, one pathway is shown in figure where the newly mapped gene products are shown in red, while the gene products that could be visualized using both the refseq and our new annotations are colored green. the blue boxes represent genes that were only found using the direct refseq blastn approach. discussion when performing microarray experiments, where the expression of tens of thousands individual genes is measured simultaneously, it is important to correctly understand the biological outcome. to achieve this, array reporters need to be annotated with the gene or gene product that they correspond with. nowadays, array producers provide annotation files containing necessary information for their arrays, such as the database identifier of the gene of which the reporter was based on, the functional name of the gene and/or the actual reporter sequence. the quality of these annotation files can differ between array producers. but even if the annotation quality is satisfactory at the moment of purchase, it is important to keep the annotation up to date. gene expression data repositories like geo and arrayexpress contain more and more experimental data. an important usage option for these repositories is to do future integrated analysis, combining different kinds of data. before this can be done, it is crucial to have adequate and up-to-date annotations. if these annotations are not updated while genome sequence databases are, we do not only lose part of the benefit of the improvements of genome annotations, but we will also lose information since older annotations do not necessarily couple to the data used in newer analytical tools. this problem often occurs when the reporters are based on unigene ids. for the incyte array used in this paper originally % of the reporters were annotated with a unigene id. a large fraction of this annotation has become useless for pathway mapping by now since the cluster ids do not longer exist in the database. this implies that it is important that databases keep track of older ids that have been used like, for instance, ensembl and unigene do, although unigene does not archive its obsolete cluster sequences. nowadays, many annotation tools are available that aim to functionally annotate a given reporter id with a specific gene ontology classification. a complementary approach is to interpret the data in a biological way by making use of pathway visualization tools, such as genmapp, metacore and/or arrayxpath ii. these tools link an element in a pathway with a specific identifier, usually by an ensembl, uniprot, unigene, entrez gene or go id. hence, to allow the biological interpretation in a pathway context, the goal should be to derive descriptions that contain the physiological most useful information for each reporter, i.e. an identifier of a protein or other gene product. some annotation methods use a blastx approach to find protein descriptions. other annotation tools do not include a blast search in their annotation procedure, but basically try to link entries from different databases to each other using a reference id. a few examples of these tools are the database for annotation, visualization and integrated discovery and the annotation builder library present in the bioconductor module of r, where the user has to supply a list of specific gene identifiers which will then be further linked towards other databases. if the gene identifier involved is correctly coupled to the array reporter, then such tools will allow us to find database reference ids in numerous genomic databases. if the gene identifier is not correctly coupled, then the biological interpretation will go astray. thus, for microarray experiments it is important to start from the beginning, i.e. by blasting the reporter sequence used on the array. during the last years much effort has been put in redefining the annotation of the affymetrix genechip arrays. a more recent paper by harbig et al. describes how they re-blasted all affymetrix human genome u133a target sequences and compared them with the annotation provided by affymetrix. their findings concluded that even for this often used chip about one third of the probe sets could be updated. furthermore, to facilitate biological understanding it is imperative to continually keep the annotation up-to-date and as accurate as possible, even if that means annotating a single reporter with more than one gene. affymetrix is aware of this problem and use an "x_at"-tag at the end of the probeset name to indicate that that probeset can detect more than one gene. but these x_at probe sets are always described with only one gene product and the other products are thus not normally included in the analysis. a related problem exists when pathway visualization tools are used. these tools do not offer a solution yet on how to visualize reporters that will hybridize strongly with more than one gene target. when researchers do know all genes that could be detected by a specific reporter, they could validate these results by pcr-based techniques using all this information. our method therefore aims to offer the end-user as much information as possible about reporter specificity: for reporters that detect more than one gene or protein product, the description, gene name and identifiers are given for all possible near-perfect alignments. it is then up to the end-user to decide if they want to include the reporter in their further analysis procedure. because of previous experiments performed on both the incyte unigene mouse i and the agilent g2519a option mouse development 44k arrays we wanted to reanalyze the data and use existing pathway tools to see if more biological information could be extracted. the existing array annotations for both arrays were problematic: the incyte array contained many annotations relating to unigene clusters that have been removed from the unigene database and are therefore no longer useful; the agilent annotation was based on the nia mouse cdna project. the reporters present on this array were derived from genomic mouse ests and clones, explaining the non-informative description for a large amount of the reporters present. because existing annotation methods using the blastn algorithm did not fulfill our criteria, we decided to develop a new method that followed a few strict criteria. uniprot was chosen as reference database because its entries are crosslinked to other databases that give relevant information for further analysis. filtering the nucleotide embl database, by only holding on to references from within a species-specific uniprot subset, resulted in a less redundant and more meaningful cleaned embl subset. a direct blastn against this cembl yields hits that are directly related to protein information. additionally, we performed a blastn against the transcripts present in the ensembl database and crosslinked the ensembl id back to end up with a uniprot id. the ensembl project plays an important role in current bioinformatics related to microarray annotation. it contains much information from genomic sequence up to the protein level. identification of the relation between a microarray reporter and an ensembl gene also allows evaluation of other aspects described or referenced in ensembl like gene splicing, variants, snps and gene orthologs. nowadays, there are many tools available that accept ensembl ids. thus, if an ensembl id contains no crosslink to uniprot, that id can still be used for continued analysis. this powerful feature makes ensembl an ideal candidate database to blast against. traditionally annotation tools often used unigene as reference database. this is in principle not wrong, but we strongly discourage this approach since it is known that after each unigene update clusters are split, joined together or removed from the database, often resulting in obsolete annotation for that reporter gene. additionally, the extra information coming from within unigene is more limited in situations where the complete genome of an organism has already been sequenced. unigene based approaches, as described above for the older incyte annotations, are still useful for reporters that were historically annotated with unigene clusters and for species where ensembl builds are not yet available. many labs still have clone collections for which they only have unigene annotations and no sequence information. after applying our annotation methods the number of reporters annotated with a uniprot or an ensembl id on either array was over 60%. this is quite high, considering the large number of reporters that originally had less-informative descriptions. whenever the original annotation also lead to a meaningful description the content of the original and our new annotation was most often identical, although our methods updated the description of % and % of these reporters for instance to a specific protein family member. for the incyte array a lot of unigene cluster ids did no longer exist, preventing further analysis relative to go terms and pathway mapping, even if the descriptions of the lost clusters was in principle correct. this loss of unigene clusters can be explained by both an improvement in the clustering algorithms used and an increase in the amounts of sequence data processed. furthermore, depositors can withdraw poor sequence data at a later time, resulting in unresolved sequences. the old incyte annotation did give annotations for a number of reporters that our new methods failed to annotate. this is primarily due to the higher alignment criteria that we applied. if these alignment criteria would be lowered, then the amount of reporters annotated would increase. for agilent, the number of probes that our methods failed to reannotate amounted to % of the total. for about one third of these lost agilent annotations the original description was non-informative. the rest originally gave specific for gene products, but could not be confirmed by our annotation methods. the cembl database contributed a considerable amount of direct protein annotations for both arrays. since thus derived annotations are more trustworthy than the ones built on the ensembl approach we preferably kept them whenever the two approaches yielded different uniprot results. we compared the results of our approach with existing approaches. first we looked at targetidentifier. this web service attempts to identify ests as full-length cdnas and to provide further functional annotation. first, the est sequences are divided into categories, based on the presence and location of a start- and stop-codon and passed through a blastx algorithm. this approach circumvents part of frame shift problems in a general blastx approach that were mentioned in the introduction as the position of start- and stop codon allows for a choice of a fixed frame. where our approach found annotations for the incyte array, targetidentifier found only uniprot id's and only of these were from the correct species. we also used a direct blast against refseq with the standard e-value of and the same filtering for at least 90% alignment that we used in all other blast procedures. the refseq approach improved the original annotation of the incyte array, but was less successful on the agilent mouse development 44k array. our methods found about 10% extra annotation on both arrays compared to the refseq approach. a much larger difference occurred when we looked for usability of this annotation in pathway mapping and gene ontology classification. this is caused by the fact that genmapp uses an ensembl based gene database. refseq protein ids are only accepted if they are linked to ensembl genes in the ensembl database itself. ensembl only includes the link between nucleotide and protein identifiers when this identity is confirmed. for most of the protein identifiers linked to refseq nm ids that are not found in ensembl this is not the case. the remaining 10% is probably caused by the refseq and ensembl builds being out of sync. to verify that the ensembl gene database is the cause of the lower acceptance of refseq ids we used biomart on build of the mus musculus ensembl database to associate the refseq dna ids, resulting from blasting against refseq, with np id's. for both the agilent and incyte reporters the number of refseq np id's found in this way corresponded exactly with the number of genes accepted in the genmapp gene database starting from the larger numbers of np identifiers found in refseq through conversion in refseq itself.. the background of this problem is that we understand biological pathways as protein functionality, and the proteins that are part of a pathway are primarily described by this protein functionality while array reporters are normally annotated with nucleotide products. the mapping of proteins to corresponding genes in databases like refseq and ensembl is still generally better than the mapping of nucleotide sequences to proteins. we tried to evaluate whether the increase in coverage comes from a decrease in specificity, meaning we annotated reporters that should not be annotated, or that we coupled the original nucleotide annotation to a protein where that is not correct. using our alignment quality criteria the actual thermodynamic hybridization conditions correspond to a one basepair mismatch in a base sequence. this essentially is what is used for the affymetrix match/mismatch method and is known to lower hybrizations to a large extend. as a result, false positives are less likely than they are in approaches where the first blast hit is used with only an e-value cut-off. database crosslinks from nucleotide databases to protein databases are often created automatically. the crosslinks from protein databases are highly curated and errors are less likely. since the initial step in our procedure uses the crosslinks from uniprot to embl it makes sense that fewer false positives will occur. to evaluate this further we compared the outcome of the annotation for reporters for our approach and the refseq and targetidentifier approaches in more detail. in general, when protein descriptions for the alternative annotations are compared they are identical. the most important differences are: 1) compared to the targetidentifier approach both our method and the refseq method found many more results. this is due to the intrinsic limitations of the targetidentifier approach which is based on blastx. also the targetidentifier approach sometimes finds hits for the wrong species which cannot be used in pathway analysis. 2) our approach was able to find protein descriptions where the refseq approach found only corresponding nucleotide sequences for 12–14% of the reporters. 3) the other way around, the refseq approach found 8–10% proteins that we only found as nucleotides. most of these could in fact be mapped to the genmapp gene database as well using the nucleotide descriptions. 4) we found 5–10% extra protein descriptions where the refseq approach found nothing. this is the most suspect group for possible false positives. for this group we checked individual reporter descriptions for the hits that we found in cembl database. these were shown to indeed contain a small number of possible false positives as they pointed to shorter bac clones. these clones were included in the cembl database since they are referred from uniprot, but the actual hit might occur away from the relevant gene sequence. while comparing the old and the new annotations we encountered some situations where a microarray reporter sequence corresponded with more than one protein: for example, agilent reporters a_66_p and a_66_p resulted in finding high quality alignment against several genes. after filtering, both reporters yielded different unique uniprot ids. for a_66_p they were respectively "histone h2a type 1", "histone h2b type 1-b " and three other family members, while for a_66_p they were "ankyrin repeat domain-containing protein 40" and "cisplatin resistance-associated overexpressed protein". in both cases the conclusion would be that the specificity of the reporters' sequence is low, allowing it to detect either more than one family member or even two functionally very different proteins. in the latter case, the reporter should be omitted from further analysis. in some specific situation both ensembl and cembl found more than different uniprot ids for agilent reporters. furthermore, the lists of proteins found showed no overlap for these reporters. direct sequence analysis and evaluation of the annotations indicated that these reporter sequences were originally unknown or derived from riken cdna. for these agilent probes, it is clear their annotation should be omitted from further analysis. this demonstrates that, next to finding the best possible description, reblasting the sequence adds an extra quality check for the reporter sequence used. after the reporter sequences are annotated, pathway visualization tools can be used to increase understanding of the biological outcome such as the genmapp program. the original unigene id's of the incyte array could be used directly for genmapp analysis, but this off course yielded poor results since many of those unigene id's are outdated. for the agilent array we verified whether the old annotations could still provide useful results by the use of methods that would couple them to ontology terms: a) by using fatigo, since the documentation explicitly claims that that webservice accepts agilent feature ids. this approach was unsuccessful, because no single reporter could be linked to a go term. b) by using the ensembl database, that contains crosslinks towards agilent reporters. in the end, this approach was not feasible, since the probe associations stored in ensembl belong to a different array type. we concluded that these methods were unable to answer our question, so a work-around was created that visualized these genes in genmapp by converting the refseq dna identifiers directly to their refseq protein counterparts. for only ~ of the ~ agilent reporters that contained a refseq reference in their original annotation were we able to find a refseq protein. this was not very different from the numbers we got when we did a direct blastn against refseq for comparison. using mappfinder, the results of our combined annotation approaches increased the amount of visualized gene products in both the local pathways and in the gene ontology classification. for both arrays, there were a few exceptions where the refseq approach was able to visualize some genes which our approach could not. the refseq approach found annotations that we did not find while we found reporters in the gene database that the refseq approach did not find. a manual reblast of these sequences against both databases indicated that the difference is mainly due to differences between the cdna sequences present in refseq and ensembl: i.e. perfect hits against refseq versus no hit at all in ensembl. apart from this, the improvement in the amount of gene products mapped was large: the number of genes that could be mapped to a local pathway increased for incyte from to to. for the agilent array a blastn against refseq found fewer sequences than the original agilent annotation, probably because of the extra alignment criteria applied. as a result it was possible to visualize a slightly smaller amount of genes in the local pathways, compared to the original, and substantially less than found with our approach. even with these relatively small changes in numbers of annotated genes the visualization power increased for a quarter of all pathways when comparing the refseq approach to the original annotation, but decreased in about half of all pathways. this increase was even stronger for the larger pathways. a full list of these individual pathway rankings are available as additional file or can be downloaded from our servers.  the usage of a high quality check for each individual reporter annotation, by requesting > 90% alignment identify, ensures that with the method described only those gene products are included in the annotation that will really hybridize. in general, the methods described in this paper increased the quality of the reporter annotations and increased the amount of annotated reporter sequences that can be visualized using pathway visualization tools. 
combining the results of both methods resulted in successful annotation of > 58% of all reporter sequences with uniprot ids on two commercial array platforms, increasing the amount of incyte reporters that could be coupled to gene ontology terms from % to % and to a local genmapp pathway from % to %. even in cases where the original reporter annotation showed the correct description the new identifiers often allowed improved pathway and gene ontology linking. the analysis of microarray experiments requires accurate and up-to-date functional annotation of the microarray reporters to optimize the interpretation of the biological processes involved. our methods increased the annotation quality of microarray reporter sequences and allowed us to visualize more reporters using pathway visualization tools. for agilent, % of the total reporters are now linked towards go nodes and % on local pathways. this paper proposes a novel method that aims to improve microarray reporter annotation by blasting the original reporter sequences against a species-specific embl subset, that was derived from and crosslinked back to the highly curated uniprot database. the resulting alignments were filtered using high quality alignment criteria and further compared with the outcome of a more traditional approach, where reporter sequences were blasted against ensembl followed by locating the corresponding protein entry for the high quality hits. pathway visualization tools are used to connect gene expression data with existing biological pathways by using specific database identifiers that link reporters with elements in the pathways.
 the analysis of microarray experiments requires accurate and up-to-date functional annotation of the microarray reporters to optimize the interpretation of the biological processes involved. pathway visualization tools are used to connect gene expression data with existing biological pathways by using specific database identifiers that link reporters with elements in the pathways.  this paper proposes a novel method that aims to improve microarray reporter annotation by blasting the original reporter sequences against a species-specific embl subset, that was derived from and crosslinked back to the highly curated uniprot database. the resulting alignments were filtered using high quality alignment criteria and further compared with the outcome of a more traditional approach, where reporter sequences were blasted against ensembl followed by locating the corresponding protein entry for the high quality hits. combining the results of both methods resulted in successful annotation of > 58% of all reporter sequences with uniprot ids on two commercial array platforms, increasing the amount of incyte reporters that could be coupled to gene ontology terms from % to % and to a local genmapp pathway from % to %. for agilent, % of the total reporters are now linked towards go nodes and % on local pathways.  our methods increased the annotation quality of microarray reporter sequences and allowed us to visualize more reporters using pathway visualization tools. even in cases where the original reporter annotation showed the correct description the new identifiers often allowed improved pathway and gene ontology linking. these methods are freely available at http://www.bigcat.unimaas.nl/public/publications/gaj_annotation/. 