BACKGROUND
transcription factor binding sites  provide a specific example of biologically functional sequence motifs that sometimes have positional preferences.
tfbss contribute substantially to the control of gene expression, and because of their biological importance, much experimental effort has been expended in identifying them.
because experimental identification is expensive, there are now many computational tools that identify tfbss as the subsequences, or "motifs", common to a set of sequences.
most tfbss correspond to short and imprecise motifs  <cit> , however, so all computational tools in a recent contest performed rather poorly in identifying known tfbss  <cit> .
although some tools have an ad hoc basis  <cit> , other tools have a basis in the calculus of probability, and can therefore immediately and systematically combine sequence with other sources of information.
most probabilistic tools align candidate subsequences and convert the nucleotide counts in the alignment columns into a position-specific score matrix .
most pssms are based on the log ratio between a motif model and a background model.
tools then identify putative motifs by maximizing the log ratio, usually with expectation maximization   <cit>  or gibbs sampling  <cit> .
experiments have shown, however, that besides common sequence motifs, tfbss also have positional preferences, as illustrated in figure  <dig>  in yeast, tfbs positions demonstrate a strong bias toward locations between  <dig> and  <dig> bases upstream of the tss  <cit> .
in e. coli, tfbs positions tend to be located between  <dig> and  <dig> bases upstream of the translation start site  <cit> .
in the words of wray et al., "for at least some regulatory elements, function constrains their position with respect to the transcriptional start site"   <cit> .
on the other hand, the trends regarding the positional preferences of tfbss appear inconsistent.
wray et al.
continue "for most transcription factors, however, binding sites lack any obvious spatial restriction relative to other feature of the locus"  <cit> .
some computational methods do exist to exploit the positional preferences of tfbss.
the first computational study using positional preferences used an empirical prior distribution of known positional information with respect to the translation start site from the e. coli genome  <cit> .
this simple method, however, is applicable only to very simple organism like e. coli.
another computational study used position to calculate p-values for candidate motifs that formed a cluster  <cit> .
the p-values were based on one particular database, however, and might not generalize reliably.
moreover, the corresponding model is not a probability model, making the systematic combination of sequence and positional information problematic.
yet another computational study modeled the positional preferences of tfbss with a uniform prior, only mentioning the possibility of a more informative prior  <cit> .
a systematic computational study to find new tfbs motifs by exploiting positional preferences applied a chi-square test to bins of positions near tsss  <cit> .
the chi-square test found one 8-letter word with significant positional preferences, the "clus1" word, tctcgcga.
the study's use of binning probably reduced the power of statistical tests, however.
shortly thereafter, in confirmation of the reduced statistical power, a systematic study of a human promoter dataset  <cit>  identified  <dig> 8-letter words with a positional preferences with respect to the tss  <cit> .
interestingly, although  <dig> of the  <dig> words appeared in the transfac database  <cit> ,  <dig> of the words did not, suggesting that tfbs positional preferences were much more pervasive than previously believed.
a later study showed that in eukaryotes the distribution of tfbss was not uniform with respect to the tss  <cit> .
a study using chromatin immunoprecipitation followed by dna hybridization  inferred tfbss within sheared dna fragments by using prior probability distributions to model positional preference  <cit> .
the model was not directed at identifying tfbss by their positional preferences with respect to genomic landmarks, however.
finally, a study applied a poisson approximation to bins of positions within promoters to identify tfbss by their positional preferences with respect to the tss  <cit> .
several studies, therefore, have examined the positional conservation of tfbss.
consequently, tfbs positional preferences are relatively well understood, particularly when compared to most non-coding dna.
very few computational tools systematically combine positional preference with sequence information, however, and to our knowledge, no general-purpose computational tools using positional information are currently available.
standard tools like meme  <cit> , alignace  <cit> , and motifsampler  <cit> , e.g., do not use positional information.
accordingly, this article evaluates the accuracy of predictions from a bayesian model combining sequence with positional information, implemented in the newest version of the tool a-glam  <cit> .
we assessed predictions from a-glam with and without the positional information, using a standard dataset of sequences with known tfbss, and were therefore able to measure the contribution of positional information to tfbs prediction accuracy.
RESULTS
results for the tss tompa dataset
the tss tompa dataset is one of two test datasets considered in this study and contains  <dig> data subsets .
table  <dig> shows an anecdotal a-glam alignment using positional information for the dataset 'hm08r' from the tss tompa dataset, which contains  <dig> sequences of length  <dig>  run in its zoops mode , a-glam returned candidate alignments with only one or zero candidate site per sequence.
in addition to sequence conservation, the alignment shows positional conservation within an interval of , much narrower than the input interval,  bp upstream of the transcription start site .
the alignment also overlapped several known sites , with a correlation coefficient of  <dig> , indicating good overlap.
a-glam predicted tfbss in  <dig> data subsets in the tss tompa data subset hm08r'.
the column "name" shows each data subset; the column "alignment", the corresponding predicted tfbs.
the start and end positions with respect to the corresponding tss are shown in the columns "start" and "end".
the columns "score" and "e-value" show bit scores and e-values that a-glam assigned to predicted tfbss.
the known binding sites in the alignment are underlined.
for tfbss predicted without positional information, e-values were immoderately small, even for incorrect predictions.
in contrast, the e-values in table  <dig> were quite moderate, perhaps because they had to reconcile conflicting constraints from different sources of information on the motifs.
alignments for more data subsets can be found in supplementary tables 1– <dig> .
we collected alignments  whose correlation coefficient  is larger than  <dig> .
the hm03r data subset does not appear in tables 1– <dig>  despite a cc of  <dig> , because the corresponding alignment had a biologically unrealistic width of  <dig> bp.
unrealistically large alignment widths are much less common for alignments with positional information than without.
in supplementary tables 2– <dig>  the alignments without positional information are omitted because they show essentially no overlap with known binding sites.
results for transfac dataset
the transfac dataset contains  <dig> data subsets.
supplementary table  <dig> contains detailed results for the input interval of .
with the addition of positional information, the ccc has improved from - <dig>  to  <dig>  with a p-value of 10- <dig> .
the ccc for transfac dataset  is smaller than for tss tompa dataset , and the positional information makes a more significant change in the ccc for the transfac dataset  than for the tss tompa dataset , probably because the transfac dataset contains  <dig> data subsets; the tss tompa dataset, only  <dig>  in the case of subtle differences, the larger transfac dataset provides more evidence, leading to smaller p-values.
cross-validation using tss tompa dataset
because we used known binding sites to estimate the hyperparameters of the model , one might suspect over-fitting.
moreover, because the distribution of locations might vary from one type of tfbs to another, the proposed model might not be appropriate for the discovery of unknown binding sites of different types of tfbss.
cross-validation addressed these issues .
over the  <dig> random partitions from tss tompa dataset, the sample average of the ccc was  <dig> ; its sample standard deviation,  <dig> ; its 90% confidence interval, ; and its range, .
the ccc for the model using sequence information alone was - <dig> .
because the ccc for sequence alone lay outside the range  of the  <dig> cccs using positional information in the 5-fold cross-validation, positional information improved prediction accuracy significantly.
the actual ccc for the model using both sequence and positional information was  <dig>  , well within the 90% confidence interval from cross-validation.
the different types of known sites have quite diverse distributions , so we expect occasional misspecification of hyperparameters η in our model .
the 5-fold cross-validation shows, however, that classification accuracy is not excessively sensitive to the hyperparameter estimation or, by extension, to the locations of the known sites.
truncation effect on sequences of test datasets
figures  <dig> and  <dig> suggest that a truncated input sequence interval of, say,  or  might incorporate positional information as well as a bayesian positional model applied to the full interval .
accordingly, in addition to the full interval , we tested  <dig> truncated intervals , , and .
the predictive accuracy, as represented by the cccs in table  <dig>  indicate that truncation on its own, without any bayesian positional modeling, improved the motif predictions.
moreover, predictive improvements due to modeling position gradually disappeared as the truncation reduced the interval to .
note, however, that positional modeling never significantly hurt the predictive accuracy, even with truncated input sequences.
the first column shows the sequence range upstream of the tss given as input to a-glam.
the change of ccc from modes with and without positional information for the tss tompa and transfac datasets is displayed in the corresponding groups of three columns.
the third column of each group shows a wilcoxon p-value, which evaluates the difference between the cccs in the previous two columns.
because not all tfbss in our datasets are known, small improvements in the ccc correspond to true improvements of unknown magnitude.
in particular, e.g., in the table, two ccc values rounded to  <dig>  have unseen decimals different enough to have a p-value of  <dig> .
to view results for individual sites in the tompa dataset, see supplementary table  <dig> .
discussion
the new version of the a-glam program   <cit>  can incorporate positional information by implementing the model from the methods section in a gibbs sampler.
a-glam already has several desirable features when predicting transcription factor binding sites .
first, it optimizes motif width automatically, without user input.
second, it reports theoretically accurate e-values for candidate tfbss.
finally, it implements a theoretically sound context-dependent markov background model, which yielded better predictions than different, ad hoc markov background models or the conventional background model of independent bases  <cit> .
with its markov background model, a rigorous statistical evaluation showed that even before the addition of positional information, a-glam's predictive accuracy was competitive with any state-of-the-art motif-finding tool  <cit> .
at the outset, we point out that all motif-finding tools have had notorious difficultly with the original tompa dataset  <cit> .
our tss tompa test dataset is even more difficult than the original tompa dataset.
its data subsets often contained fewer sequences than the corresponding original tompa subset.
moreover, our sequences were on average longer than the corresponding original tompa sequence.
thus, conventional motif-finding tools should perform more weakly on our tss tompa test dataset than on the original tompa dataset.
the bayesian model in this paper combines sequence and positional information to predict putative tfbss.
its implementation in a-glam permits users either to accept our default hyperparameters η for the prior distribution or to select their own.
although complete flexibility in the selection of hyperparameters can permit inappropriate or excessively aggressive choices, extensive cross-validation showed that the usual priors place mild restrictions on the predictions, so the model is very robust against misspecification of its hyperparameters or, by extension, to the locations of known sites.
in other words, the prior does not dictate the alignment; instead, it loosely guides the alignment and permits the data to "speak for themselves".
if motifs do not cluster by position, a-glam might therefore still find motifs sharing sequence but not position.
we therefore make the following recommendation to users: in the absence of a strong reason to the contrary, they should accept a-glam's default hyperparameters.
to use positional information to find biologically active sites, a-glam's positional model requires the input sequences to be anchored on a genomic landmark, e.g., to find tfbss, the model might be anchored to tsss.
because a single gene might correspond to several alternative tsss  <cit> , however, tss multiplicity might initially appear to cause problems.
moreover, the tss itself can have either "sharp" or "broad" positional preference within a promoter  <cit> .
variability of the tss position within a promoter reduces the positional information available to a-glam, possibly explaining the uneven improvement in prediction across our data subsets.
a-glam's statistical model examines sequence as well as positional information, however, so it retains robustness against a mild misspecification of the tss, say, within a few hundred bases of the true position, so alternative tsss or tsss with a typical broad positional distribution are unlikely to degrade predictions seriously when positional information is used.
a-glam's users should note, however, if a tss is specified, e.g., a kilobase away from the relevant position, positional information might severely distract a-glam from finding the desired tfbss.
on the other hand, however, different positions relative to the tss containing exactly the same sequence have long been known to be associated with different tfbs biological functions  <cit> ; in other cases, they might also be associated with alternative tsss or tsss with a broad positional distribution.
up to now, because computational studies of positional control of transcription have had to rely on ad hoc methods, a-glam now has a unique potential among general motif-prediction tools.
even if two functionally different sets of tfbss have similar motifs, a-glam can differentiate them by position alone and report the two sets separately.
it would be very interesting if someone using a-glam identified two sets of tfbss of similar sequence corresponding to two different functionalities or tsss.
the sequences in our study used the upstream positions from - <dig> to  <dig> bp relative to the tss to evaluate a-glam's accuracy in predicting tsss.
because our purpose in this article was to evaluate a-glam's ability to find biologically active sequence motifs in general, there is no scientific reason not to use the 3' utr region as a "genomic anchor" to identify nearby regulatory elements.
a similar statement applies to any set of regulatory elements  around any genomic landmark .
indeed, if its main purpose was not evaluation of the predictive accuracy of a-glam's positional model, this article could have restricted its input sequences to intervals downstream of the tss, e.g.,  <cit>  bp instead.
with the tss still providing the genomic anchor, a-glam could have searched for motifs associated with, e.g., 5' utrs or translation start sites, which are usually within a few hundred base pairs downstream of a tss.
thus, positional restrictions on the input sequence could focus a-glam's search on sequence motifs with different biological functions.
in practice, however, restricting the input interval requires great care.
unlike the tfbss in our test datasets, many sequence motifs have poorly characterized distributions.
on one hand, excessively stringent truncation of the input interval to, say,  would probably have removed many tfbss from consideration in our study.
on the other hand, positional modeling generally improved the accuracy of motif prediction, never hurting it significantly, even when input sequences were truncated.
in the search for novel sequence motifs, therefore, we recommend that the use of bayesian positional modeling on an input sequence whose length is generous  relative to the locations of known motifs.
since the previous study showed that a-glam is one of the top performers among existing tools for de novo tfbs discovery  <cit> , we believe that a-glam now easily outperforms its competitors whenever positional information is available and relevant.
"positional genomics" exploits the information provided by genomic landmarks , yielding a "poor man's alignment", even when the precise sequence alignments are unavailable.
given the power of comparative genomics, which depends on accurate alignments, positional genomics presents many interesting possibilities.
CONCLUSIONS
we proposed a bayesian model for incorporating positional preference of tfbs with respect to a genomic landmark, e.g., a tss.
the results on our test datasets show that a positional model can produce statistically significant improvements in the accuracy of motif prediction.
our cross-validation study shows that the prior distribution of our positional model is robust against mild misspecification of its parameters.
our study of truncated input sequences indicates that the positional model provides a superior and more robust strategy than sequence truncation, especially when the positional preferences of sequence motifs are not well characterized.
availability
the a-glam program and all datasets relevant to this article can be found online  <cit> .
project name: a-glam  <dig>
project home page:
operating system: linux
programming language: c++
licence: no license required.

 transcription factor binding sites provide a specific example of biologically functional sequence motifs that sometimes have positional preferences. tfbss contribute substantially to the control of gene expression, and because of their biological importance, much experimental effort has been expended in identifying them. because experimental identification is expensive, there are now many computational tools that identify tfbss as the subsequences, or "motifs", common to a set of sequences. most tfbss correspond to short and imprecise motifs, however, so all computational tools in a recent contest performed rather poorly in identifying known tfbss. although some tools have an ad hoc basis, other tools have a basis in the calculus of probability, and can therefore immediately and systematically combine sequence with other sources of information. most probabilistic tools align candidate subsequences and convert the nucleotide counts in the alignment columns into a position-specific score matrix. most pssms are based on the log ratio between a motif model and a background model. tools then identify putative motifs by maximizing the log ratio, usually with expectation maximization or gibbs sampling. experiments have shown, however, that besides common sequence motifs, tfbss also have positional preferences, as illustrated in figure in yeast, tfbs positions demonstrate a strong bias toward locations between and bases upstream of the tss. in e. coli, tfbs positions tend to be located between and bases upstream of the translation start site. in the words of wray et al., "for at least some regulatory elements, function constrains their position with respect to the transcriptional start site". on the other hand, the trends regarding the positional preferences of tfbss appear inconsistent. wray et al. continue "for most transcription factors, however, binding sites lack any obvious spatial restriction relative to other feature of the locus". some computational methods do exist to exploit the positional preferences of tfbss. the first computational study using positional preferences used an empirical prior distribution of known positional information with respect to the translation start site from the e. coli genome. this simple method, however, is applicable only to very simple organism like e. coli. another computational study used position to calculate p-values for candidate motifs that formed a cluster. the p-values were based on one particular database, however, and might not generalize reliably. moreover, the corresponding model is not a probability model, making the systematic combination of sequence and positional information problematic. yet another computational study modeled the positional preferences of tfbss with a uniform prior, only mentioning the possibility of a more informative prior. a systematic computational study to find new tfbs motifs by exploiting positional preferences applied a chi-square test to bins of positions near tsss. the chi-square test found one 8-letter word with significant positional preferences, the "clus1" word, tctcgcga. the study's use of binning probably reduced the power of statistical tests, however. shortly thereafter, in confirmation of the reduced statistical power, a systematic study of a human promoter dataset identified 8-letter words with a positional preferences with respect to the tss. interestingly, although of the words appeared in the transfac database, of the words did not, suggesting that tfbs positional preferences were much more pervasive than previously believed. a later study showed that in eukaryotes the distribution of tfbss was not uniform with respect to the tss. a study using chromatin immunoprecipitation followed by dna hybridization inferred tfbss within sheared dna fragments by using prior probability distributions to model positional preference. the model was not directed at identifying tfbss by their positional preferences with respect to genomic landmarks, however. finally, a study applied a poisson approximation to bins of positions within promoters to identify tfbss by their positional preferences with respect to the tss. several studies, therefore, have examined the positional conservation of tfbss. consequently, tfbs positional preferences are relatively well understood, particularly when compared to most non-coding dna. very few computational tools systematically combine positional preference with sequence information, however, and to our knowledge, no general-purpose computational tools using positional information are currently available. standard tools like meme, alignace, and motifsampler, e.g., do not use positional information. accordingly, this article evaluates the accuracy of predictions from a bayesian model combining sequence with positional information, implemented in the newest version of the tool a-glam. we assessed predictions from a-glam with and without the positional information, using a standard dataset of sequences with known tfbss, and were therefore able to measure the contribution of positional information to tfbs prediction accuracy.  results for the tss tompa dataset the tss tompa dataset is one of two test datasets considered in this study and contains data subsets. table shows an anecdotal a-glam alignment using positional information for the dataset'hm08r' from the tss tompa dataset, which contains sequences of length run in its zoops mode, a-glam returned candidate alignments with only one or zero candidate site per sequence. in addition to sequence conservation, the alignment shows positional conservation within an interval of, much narrower than the input interval, bp upstream of the transcription start site. the alignment also overlapped several known sites, with a correlation coefficient of, indicating good overlap. a-glam predicted tfbss in data subsets in the tss tompa data subset hm08r'. the column "name" shows each data subset; the column "alignment", the corresponding predicted tfbs. the start and end positions with respect to the corresponding tss are shown in the columns "start" and "end". the columns "score" and "e-value" show bit scores and e-values that a-glam assigned to predicted tfbss. the known binding sites in the alignment are underlined. for tfbss predicted without positional information, e-values were immoderately small, even for incorrect predictions. in contrast, the e-values in table were quite moderate, perhaps because they had to reconcile conflicting constraints from different sources of information on the motifs. alignments for more data subsets can be found in supplementary tables 1–. we collected alignments whose correlation coefficient is larger than. the hm03r data subset does not appear in tables 1– despite a cc of, because the corresponding alignment had a biologically unrealistic width of bp. unrealistically large alignment widths are much less common for alignments with positional information than without. in supplementary tables 2– the alignments without positional information are omitted because they show essentially no overlap with known binding sites. results for transfac dataset the transfac dataset contains data subsets. supplementary table contains detailed results for the input interval of. with the addition of positional information, the ccc has improved from - to with a p-value of 10-. the ccc for transfac dataset is smaller than for tss tompa dataset, and the positional information makes a more significant change in the ccc for the transfac dataset than for the tss tompa dataset, probably because the transfac dataset contains data subsets; the tss tompa dataset, only in the case of subtle differences, the larger transfac dataset provides more evidence, leading to smaller p-values. cross-validation using tss tompa dataset because we used known binding sites to estimate the hyperparameters of the model, one might suspect over-fitting. moreover, because the distribution of locations might vary from one type of tfbs to another, the proposed model might not be appropriate for the discovery of unknown binding sites of different types of tfbss. cross-validation addressed these issues. over the random partitions from tss tompa dataset, the sample average of the ccc was; its sample standard deviation,; its 90% confidence interval,; and its range,. the ccc for the model using sequence information alone was -. because the ccc for sequence alone lay outside the range of the cccs using positional information in the 5-fold cross-validation, positional information improved prediction accuracy significantly. the actual ccc for the model using both sequence and positional information was, well within the 90% confidence interval from cross-validation. the different types of known sites have quite diverse distributions, so we expect occasional misspecification of hyperparameters η in our model. the 5-fold cross-validation shows, however, that classification accuracy is not excessively sensitive to the hyperparameter estimation or, by extension, to the locations of the known sites. truncation effect on sequences of test datasets figures and suggest that a truncated input sequence interval of, say, or might incorporate positional information as well as a bayesian positional model applied to the full interval. accordingly, in addition to the full interval, we tested truncated intervals,, and. the predictive accuracy, as represented by the cccs in table indicate that truncation on its own, without any bayesian positional modeling, improved the motif predictions. moreover, predictive improvements due to modeling position gradually disappeared as the truncation reduced the interval to. note, however, that positional modeling never significantly hurt the predictive accuracy, even with truncated input sequences. the first column shows the sequence range upstream of the tss given as input to a-glam. the change of ccc from modes with and without positional information for the tss tompa and transfac datasets is displayed in the corresponding groups of three columns. the third column of each group shows a wilcoxon p-value, which evaluates the difference between the cccs in the previous two columns. because not all tfbss in our datasets are known, small improvements in the ccc correspond to true improvements of unknown magnitude. in particular, e.g., in the table, two ccc values rounded to have unseen decimals different enough to have a p-value of. to view results for individual sites in the tompa dataset, see supplementary table. discussion the new version of the a-glam program can incorporate positional information by implementing the model from the methods section in a gibbs sampler. a-glam already has several desirable features when predicting transcription factor binding sites. first, it optimizes motif width automatically, without user input. second, it reports theoretically accurate e-values for candidate tfbss. finally, it implements a theoretically sound context-dependent markov background model, which yielded better predictions than different, ad hoc markov background models or the conventional background model of independent bases. with its markov background model, a rigorous statistical evaluation showed that even before the addition of positional information, a-glam's predictive accuracy was competitive with any state-of-the-art motif-finding tool. at the outset, we point out that all motif-finding tools have had notorious difficultly with the original tompa dataset. our tss tompa test dataset is even more difficult than the original tompa dataset. its data subsets often contained fewer sequences than the corresponding original tompa subset. moreover, our sequences were on average longer than the corresponding original tompa sequence. thus, conventional motif-finding tools should perform more weakly on our tss tompa test dataset than on the original tompa dataset. the bayesian model in this paper combines sequence and positional information to predict putative tfbss. its implementation in a-glam permits users either to accept our default hyperparameters η for the prior distribution or to select their own. although complete flexibility in the selection of hyperparameters can permit inappropriate or excessively aggressive choices, extensive cross-validation showed that the usual priors place mild restrictions on the predictions, so the model is very robust against misspecification of its hyperparameters or, by extension, to the locations of known sites. in other words, the prior does not dictate the alignment; instead, it loosely guides the alignment and permits the data to "speak for themselves". if motifs do not cluster by position, a-glam might therefore still find motifs sharing sequence but not position. we therefore make the following recommendation to users: in the absence of a strong reason to the contrary, they should accept a-glam's default hyperparameters. to use positional information to find biologically active sites, a-glam's positional model requires the input sequences to be anchored on a genomic landmark, e.g., to find tfbss, the model might be anchored to tsss. because a single gene might correspond to several alternative tsss, however, tss multiplicity might initially appear to cause problems. moreover, the tss itself can have either "sharp" or "broad" positional preference within a promoter. variability of the tss position within a promoter reduces the positional information available to a-glam, possibly explaining the uneven improvement in prediction across our data subsets. a-glam's statistical model examines sequence as well as positional information, however, so it retains robustness against a mild misspecification of the tss, say, within a few hundred bases of the true position, so alternative tsss or tsss with a typical broad positional distribution are unlikely to degrade predictions seriously when positional information is used. a-glam's users should note, however, if a tss is specified, e.g., a kilobase away from the relevant position, positional information might severely distract a-glam from finding the desired tfbss. on the other hand, however, different positions relative to the tss containing exactly the same sequence have long been known to be associated with different tfbs biological functions; in other cases, they might also be associated with alternative tsss or tsss with a broad positional distribution. up to now, because computational studies of positional control of transcription have had to rely on ad hoc methods, a-glam now has a unique potential among general motif-prediction tools. even if two functionally different sets of tfbss have similar motifs, a-glam can differentiate them by position alone and report the two sets separately. it would be very interesting if someone using a-glam identified two sets of tfbss of similar sequence corresponding to two different functionalities or tsss. the sequences in our study used the upstream positions from - to bp relative to the tss to evaluate a-glam's accuracy in predicting tsss. because our purpose in this article was to evaluate a-glam's ability to find biologically active sequence motifs in general, there is no scientific reason not to use the 3' utr region as a "genomic anchor" to identify nearby regulatory elements. a similar statement applies to any set of regulatory elements around any genomic landmark. indeed, if its main purpose was not evaluation of the predictive accuracy of a-glam's positional model, this article could have restricted its input sequences to intervals downstream of the tss, e.g., bp instead. with the tss still providing the genomic anchor, a-glam could have searched for motifs associated with, e.g., 5' utrs or translation start sites, which are usually within a few hundred base pairs downstream of a tss. thus, positional restrictions on the input sequence could focus a-glam's search on sequence motifs with different biological functions. in practice, however, restricting the input interval requires great care. unlike the tfbss in our test datasets, many sequence motifs have poorly characterized distributions. on one hand, excessively stringent truncation of the input interval to, say, would probably have removed many tfbss from consideration in our study. on the other hand, positional modeling generally improved the accuracy of motif prediction, never hurting it significantly, even when input sequences were truncated. in the search for novel sequence motifs, therefore, we recommend that the use of bayesian positional modeling on an input sequence whose length is generous relative to the locations of known motifs. since the previous study showed that a-glam is one of the top performers among existing tools for de novo tfbs discovery, we believe that a-glam now easily outperforms its competitors whenever positional information is available and relevant. "positional genomics" exploits the information provided by genomic landmarks, yielding a "poor man's alignment", even when the precise sequence alignments are unavailable. given the power of comparative genomics, which depends on accurate alignments, positional genomics presents many interesting possibilities.  we proposed a bayesian model for incorporating positional preference of tfbs with respect to a genomic landmark, e.g., a tss. the results on our test datasets show that a positional model can produce statistically significant improvements in the accuracy of motif prediction. our cross-validation study shows that the prior distribution of our positional model is robust against mild misspecification of its parameters. our study of truncated input sequences indicates that the positional model provides a superior and more robust strategy than sequence truncation, especially when the positional preferences of sequence motifs are not well characterized. availability the a-glam program and all datasets relevant to this article can be found online. project name: a-glam project home page: operating system: linux programming language: c++ licence: no license required. 
as expected, when sequences in the datasets were successively truncated to the intervals, and, positional information aided motif prediction less and less, but never hurt it significantly. for example, many known transcription factor binding sites occur within an interval bases upstream of a transcription start site. biologically active sequence motifs often have positional preferences with respect to a genomic landmark. although sequence truncation is a viable strategy when searching for biologically active motifs with a positional preference, a probabilistic model generally provides a superior and more robust strategy, particularly when the sequence motifs' positional preferences are not well characterized. although some programs for identifying sequence motifs exploit positional information, most of them model it only implicitly and with ad hoc methods, making them unsuitable for general motif searches. a-glam, a user-friendly computer program for identifying sequence motifs, now incorporates a bayesian model systematically combining sequence and positional information. a rigorous statistical analysis showed that positional information significantly improved the prediction of sequence motifs, and an extensive cross-validation study showed that a-glam's model was robust against mild misspecification of its parameters. a-glam's predictions with and without positional information were compared on two human tfbs datasets, each containing sequences corresponding to the interval bases upstream of a known tss.
 biologically active sequence motifs often have positional preferences with respect to a genomic landmark. for example, many known transcription factor binding sites occur within an interval bases upstream of a transcription start site. although some programs for identifying sequence motifs exploit positional information, most of them model it only implicitly and with ad hoc methods, making them unsuitable for general motif searches.  a-glam, a user-friendly computer program for identifying sequence motifs, now incorporates a bayesian model systematically combining sequence and positional information. a-glam's predictions with and without positional information were compared on two human tfbs datasets, each containing sequences corresponding to the interval bases upstream of a known tss. a rigorous statistical analysis showed that positional information significantly improved the prediction of sequence motifs, and an extensive cross-validation study showed that a-glam's model was robust against mild misspecification of its parameters. as expected, when sequences in the datasets were successively truncated to the intervals, and, positional information aided motif prediction less and less, but never hurt it significantly.  although sequence truncation is a viable strategy when searching for biologically active motifs with a positional preference, a probabilistic model generally provides a superior and more robust strategy, particularly when the sequence motifs' positional preferences are not well characterized. 