BACKGROUND
dna microarray is one of the most powerful and versatile tools for post-genomic research  <cit> .
after the initial success with cdna and pcr product-based microarrays, application of long oligonucleotides became widely used in "spotted" dna microarray technology in the last five years  <cit> .
from the beginning it became clear that the design of the oligonucleotide probes requires special attention.
under a single stringency condition, hybridization specificity and efficiency of all oligonucleotides must be globally maximized across the entire array.
thus for the selection of the optimal oligonucleotide candidates, four major parameters are being evaluated:  uniqueness which analyzes other possible cross-hybridization targets in the genome;  sequence complexity which evaluates the presence of short nucleotide repeats;  melting temperature  or gc content which ensures a uniform hybridization efficiency across the microarray; and  level of internal secondary structures which helps to avoid all possible self-binding interference with the specific target hybridization.
in principle each of these properties can be calculated individually for every potential oligonucleotide candidate, however, the main challenge that remains is to derive a selection strategy that combines these parameters and selects the most optimal oligonucleotide representative for a given genetic locus/gene.
all currently available programs for long oligonucleotide microarray design utilize different parameters: the binding energy or blast-based score to alternative targets to evaluate uniqueness, the gc content or tm to estimate hybridization stringency, the reverse smith-waterman score or free energy to evaluate levels of secondary structure and various types of complexity coefficients to evaluate the presence of short nucleotide repeats in each oligonucleotide element  <cit> .
typically these programs select one or more oligonucleotide representatives of a gene using various systems of cutoff-based filters.
for example arrayoligoselector creates an intersection of oligonucleotides that pass parameter-based cutoffs for uniqueness, self-binding and sequence complexity.
the intersection candidate list is then passed on to the gc filter and subsequently the final representative are selected using a 3' proximity criteria  <cit> .
the cutoff based algorithms provide a powerful approach to select dna microarray oligonucleotide sets and were successfully used to design dna microarrays for a large number of species  <cit> .
the use of these algorithms is, however, not completely optimal for genomes with high abundance of repetitive sequences and large fluctuations of gc content.
to accommodate such genomic sequences, the methods must relax the parameter filter adjustments.
the wide "opening" of the cutoff filters can cause selection of suboptimal oligonucleotides for a significant number of genes, due to the fact that all oligonucleotides that pass a particular filter are treated as equal by the subsequent steps, disregarding their subtle diversity within the filtered interval of the parameter .
to overcome these shortcomings new algorithms which incorporate optimization strategies of oligonucleotide parameters were developed including oligodesign  <cit>  and commoligo  <cit> .
oligodesign was developed specifically for the design of the locked nucleic acid  microarray platform which takes advantage of the improved nucleic on-chip capture sensitivity of the lna substitute mixmer oligonucleotides.
design of these specialized probes requires careful optimizations of the hybridization specificity and efficiency for each probe.
for this purpose, oligodesign uses an extensive fuzzification process derived from neural network approaches to ensure the optimal performance of this highly specialized microarray platform  <cit> .
similar to the fuzzy logic approach, commoligo uses a piece-wise linear function to select optimal oligonucleotides via a user configurable iterative process  <cit> .
both of these methods represented a step in the right direction, recognizing the need for parallel optimization of all used parameters and elimination of cutoffs that cause information loss.
at its presently available implementation, however, both oligodesign and commoligo utilize complex and computer-time consuming processes that render them unsuitable for high throughput applications.
nevertheless both methods have been useful for design of focused "miniarrays" which typically contain smaller numbers of genes e.g.
<dig> stress response and toxicological markers from caenorhabditis elegans  <cit>  or microarrays for relatively small genomes such as methanoccocus maripaludis with  <dig> genes  <cit> .
here we present a novel program named oligorankpick that is inspired by the aforementioned parameter optimization approaches and it is suitable for the design of gene specific long oligonucleotide probes for genomes of all sizes.
the final decision making process is based on a weighted rank-sum strategy which significantly streamlines the entire computation process.
this complete eliminates all cutoff based filters, thereby significantly improves the quality of the resulting microarray oligonucleotide design.
moreover, the weighted rank-sum approach enables us to implement an integer weighted linear function to automatically optimize the oligonucleotide parameters for each gene individually.
finally to demonstrate the utility of oligorankpick we design, assemble and verify a new version of p. falciparum microarray which comprises of  <dig> oligonucleotides representing  <dig> genes.
RESULTS
algorithm overview
rank transformation
several previous methods have been introduced to normalize oligonucleotide parameter scores including a piece-wise, and sigmoid function  <cit> .
in our approach the transformations of score values calculated for each parameter into rank numbers allows us to uniformly assesses and adjust the contribution of different parameters for the optimal oligonucleotide selection.
for all coding sequences of the test genome  there is a complete agreement between the overall profiles of the scores and the converted ranks in each parameter .
this indicates that the rank transformation does not affect the oligonucleotide status in the oligonucleotide set, and that the rank transformation has an equal power of parameter comparisons to the original scores.
optimizations of weight sets
the challenge of deriving weight values that will select an optimal oligonucleotide in the rank-sum strategy is two-fold:  the weight coefficients should correspond to the relative contribution of each parameter to the oligonucleotide performance during the microarray hybridization ;  the weight set optimization should provide sufficient flexibility to accommodate the variable nature of the primary structure along the genome.
for this purpose we aimed to develop a strategy that optimizes the weight set for each gene individually by considering broader intervals of weight coefficients rather then a single target value .
to derive and subsequently evaluate this strategy, we used the p. falciparum genome which is characterized by large fluctuations of gc content, and an abundance of repetitive sequences and large highly homologous gene families  <cit> .
in the first step we calculated the top oligonucleotide for all plasmodium genes using  <dig> different weight sets.
these sets originate from all combinations of four weight intervals: wblast= <cit> , wgc= <cit> , wsw= <cit> , wlz= <cit> .
the broader intervals for uniqueness and cg content weight coefficients are intended to allow higher impact of these parameters on the final oligonucleotide selection.
these adjustments are based on our previous observations, indicating that variations in gc content and uniqueness have a greater effect on specificity and efficiency of microarray hybridization compared to the other parameters  <cit>  and bozdech et al unpublished data).
the implementation of weight interval optimization strategy  then facilitates the gene specific optimizations of the oligonucleotide selection with respect to the sequence properties of a particular gene.
previous studies suggest that 40% identity of continuous match is an upper limit for possible microarray cross-hybridization signal for oligonucleotides between  <dig> and  <dig> nucleotides   <cit> .
for the gc content a 5% deviation  will result in a fluctuation in tm of 4°c which provides a theoretical upper limit for microarray hybridization stringency .
we utilized these two rules ) as criteria to evaluate the quality of oligonucleotide selection .
it is important to emphasize that these criteria are not implemented as cutoff values in the oligorankpick algorithm but as quality monitoring criteria to evaluate the selection process utilizing varying weight sets.
first  <dig> oligonucleotide sets of the p. falciparum genome were generated using all combinations of the initial weight values.
then the number of oligonucleotides that passed the both rules was calculated .
in the uniqueness weight simulations the number of oligonucleotides outside of the limit criteria gradually plateaus for the uniqueness weights  greater than  <dig>  for wblast values greater then  <dig> there were essentially no further oligonucleotide attritions regardless of the other weight values .
by manual inspection we observed that the non-unique oligonucleotides  originate mostly from duplicated genes for which no unique oligonucleotides could be selected.
the gc content modeling exhibits essentially identical tendencies with the vast majority of oligonucleotides that were found outside of the gc range originating from genes with an extremely high at content .
taken together both gc content and uniqueness weight values bellow  <dig> do not generate enough power to maximize these parameters while values greater then  <dig> do not improve the numbers of acceptable oligonucleotides due to the natural properties of the genome.
thus in order to streamline the oligonucleotide design for the following studies, we built a limited weight pool composed of uniqueness weights and gc content from  <dig> to  <dig>  sw weight is  <dig> or  <dig>  and lz is defined as  <dig>  this scheme creates new  <dig> optimized sets  which can be used effective oligonucleotide selection.
based on these simulations, we expect that the  <dig> weight sets will provide sufficient flexibility and efficiency to optimize all four parameters, while preserving the higher importance of the gc content and uniqueness.
in summary the oligorankpick algorithm is capable of designing microarray oligonucleotide sets avoiding any arbitrary cutoff implemented directly in the selection algorithm.
instead this algorithm incorporates various empirical and theoretical boundaries via target values to optimize the simulation step during which specific weight intervals are determined.
comparison with other programs
to compare the performance of oligorankpick with other publicly available programs, we designed three theoretical microarray oligonucleotide sets for the p. falciparum, s. cerevisiae and e. coli.
we selected three programs, arrayoligoselector  <cit> , oligopicker  <cit>  and oligoarray  <dig>   <cit> .
for the intended designs we chose the oligonucleotide length to be  <dig> nt and the gc content  <dig> %  for p. falciparum, 40%  for s. cerevisiae and 45% for e. coli .
the theoretical oligonucleotide sets were designed using the publicly available sequence data and the selection algorithms with default settings.
figure  <dig> summarizes the parameter distributions of the uniqueness scores  plotted against gc content.
overall these contour plots illustrate that comparing to the three publicly available programs, oligorankpick provides significant improvements for the design of yeast, e. coli and p. falciparum microarray .
the most striking improvements were, however, observed in the design of the p. falciparum microarray.
for this genome the blast scores and the gc content of the oligonucleotides designed by oligorankpick exhibit a greater convergence to a small region in the desired area  compared to oligonucleotides designed by the three other programs .
similar convergence is observed for the sw and lz scores .
to further demonstrate the convergence of the oligonucleotide parameters we calculated a mean distance for each parameter distribution to its desired  value and also to the average value within the parameter distribution .
in all cases the oligorankpick produced the smallest mean distances and thus tighter distribution of the oligonucleotide parameters.
the only exception is the lower mean distance of the cg content from its mean value in the yeast set designed by oligopicker.
detailed inspection of these results indicated that the low mean distance is due to extensive filtering implemented by this program .
for each of the theoretical microarray dataset we also calculate the average weight score  which is directly related to the oligonucleotide quality with respect to the oligonucleotide parameters .
the smaller aws that are consistently observed for the oligorankpick generated oligonucleotide sets compared to the three other programs further indicate the optimization power of oligorankpick.
*arrayoligoselector  <dig> .4; oligopicker; oligoarray  <dig> .
a: oligonucleotide number selected by the program; b: good oligonucleotide number based on blast score of non-target  and gc content .
& percentage of good quality oligonucleotide to total selected oligonucleotide .
ξ five rejected coding sequences are less than  <dig> bp.
ζ only one rejected sequence is yjr151w-a .
one of the unique features of the p. falciparum genome is the presence of several large highly homologous gene families whose role has been implicated in the antigenic variation including var , rifin  and stevor  <cit> .
table  <dig> indicates the number of unique oligonucleotides designed by all the four programs for these genes.
oligorankpick was capable of designing unique oligonucleotides for  <dig> genes  of total  <dig> genes which by far exceeded the performance of the three other algorithms.
design of a gene specific dna microarray for p. falciparum
*arrayoligoselector  <dig> .4; oligopicker; oligoarray  <dig> .
a: oligonucleotide number selected by the program; b: accepted oligonucleotide number based on blast score of non-target .
in the final step we applied oligorankpick to design a gene specific dna microarray for the p. falciparum genome  that can be used for functional genomic studies of this important human pathogen.
for this design we wished to increase the oligonucleotide coverage for longer open reading frames and thus we fragmented each coding sequence using the fragmentation.pl script as follows: sequences smaller than  <dig> kb were kept as one fragment; sequences between  <dig> kb and  <dig> kb were split evenly into two fragments, sequences larger then  <dig> kb were split into n fragments  when: kb < gene size > kb.
the fragmentation step generated  <dig> microarray element fragments  from  <dig> cds.
a single oligonucleotide was designed for each mef which resulted in one oligonucleotide per  <dig> bp on average for all p. falciparum coding sequences.
although the median gc content of all  <dig> nt oligonucleotide windows in the p. falciparum coding sequences is  <dig> %  for higher specificity and efficiency of microarray hybridization, we selected oligonucleotides with a gc content of  <dig> % .
oligorankpick successfully designed  <dig> oligonucleotides representing all predicted p. falciparum genes with an average of  <dig>  oligonucleotides per protein coding sequence .
figure 4b summarizes the gc content distribution suggesting that oligorankpick can identify optimal oligonucleotide elements with gc content significantly distant from the average gc content in the genome.
astonishingly  <dig> % of the designed oligonucleotides had the desired gc content of  <dig> % .
to evaluate the level of uniqueness of the designed oligonucleotides we used the identical quality control criteria used for the weight optimization strategy which is consistent with previously established conditions of optimal microarray hybridization performance .
in total  <dig>  oligonucleotides passed the uniqueness criteria and  <dig> oligonucleotides were found to be in the range of 5% deviation from the gc content target value  .
there are  <dig>  oligonucleotides meeting both criteria while only  <dig> oligonucleotides  were outside of the ± 5% gc content interval and  <dig> oligonucleotides  were not unique in the genome.
manual inspections of the mefs represented by these oligonucleotides indicated that no suitable  <dig> nt window exists within these dna fragments.
the  <dig> non-unique oligonucleotides represented  <dig> genes  from which  <dig> genes belong to the large multigenic gene families, var,rifin and stevor.
pair-wise sequence homology analysis of these genes revealed that these genes do not contain any  <dig> nt window that shares less than 40% homology with any other member of the corresponding gene family and thus no unique oligonucleotide could be selected by any conceivable strategy.
interestingly for the remaining  <dig>  members of these families a specific oligonucleotide was selected which further demonstrates the power of oligorankpick for microarray design.
transcriptome analysis of the trophozoite and schizont stages of p. falciparum
although all parameters of the oligonucleotide microarray sets designed by oligorankpick indicate their high quality, the ultimate evidence for their functionality can be provided only by physical microarray experiments.
for this purpose we have synthesized all the  <dig> oligonucleotides for the p. falciparum genome-wide microarray and spotted these onto polylysine-coated microscopic slides as previously described  <cit> .
using these microarrays we compare the global mrna patterns between two developmental stages of the p. falciparum intraerythrocytic development, trophozoite and schizont.
all experimental procedures were carried out as previously described  <cit>  and the complete results for three replicates of the microarray hybridizations are available in the supplementary data.
the p. falciparum genome sequence reference strain 3d <dig> was used for this analysis.
total  <dig> genes were found to be expressed in at least one of the studied developmental stages in three replicates of microarray hybridization.
from these  <dig> and  <dig> mrna transcripts exhibited at least 2-fold higher abundance in the trophozoite and the schizont stage, respectively .
in order to assess the fidelity of the obtained results we wish to compare this data to previously published transcriptome analyses of the p. falciparum intraerythrocytic developmental cycle .
these include the idc transcriptome analyzed by the previous version of a long oligonulceotide microarray  comprised of  <dig> genes  <cit> , and a high density short oligonucleotide affymetrix microarray dataset  comprised of  <dig> genes with stage specific transcription  <cit> .
all genes present in both lom-idc and hdso-affymetrix transcriptomes were represented on the new p. falciparum microarray and yielded a hybridization signal in at least two of the three microarray replicates.
to compare the stage specificity of the gene expression we select genes which exhibited > 3-fold change in mrna abundance between trophozoite and schizonts detected in at least two  replicates .
using these criteria we classify  <dig> genes as trophozoite specific and  <dig> genes as schizont specific.
the transcriptome data comparisons, summarized in table  <dig>  indicate high correlations between the transcriptome data and the new microarray dataset with  <dig> – <dig> % of overlapping genes exhibiting identical stage specificity in their mrna levels.
there were only a small number of genes  for which the new expression results did not correlate with the previously published data.
these discrepancies are likely caused by subtle differences in parasite culture synchronicity and stage representation between our culturing system and the systems used for the previous transcriptome analyses.
*genes with peak expression before and after  <dig> hours post invasion are classified as trophozoite and schizont specific, respectively
** genes with higher expression levels in late ring and early and late trophozoites compared to early and late schizonts are classified as trophozoite specific and vice versa
to further validate the performance of the designed p. falciparum microarray quantitative real-time rt-pcr was used to measure relative mrna abundance between trophozoite and schizont stage for  <dig> selected genes.
for this we chose genes for which only oligorankpick designed a "good quality" microarray element while the three tested publicly available programs did not yield a suitable oligonucleotide element.
these include two paralogous histone <dig>  five members of the variable surface antigen gene families , centrin, and two genes encoding highly homologous hypothetical proteins.
figure 5a shows good correlations between the rt-pcr results and microarray hybridization data which demonstrate the robust performance of the newly designed microarray for analyses of mrna abundance in p. falciparum.
detail sequence analyses revealed that each of the  <dig> selected genes contains only a small window of unique sequence while the majority of the gene is highly homologous to at least one other locus in the genome.
one of the example is a pair of highly homologous genes encoding histone <dig>  and its homologue histone <dig>   .
this high homology is likely the main obstacle for designing a specific oligonucleotide and it is the reason why no transcription data have been obtained by the previously reported transcriptome analyses.
despite this oligorankpick selected specific oligonucleotides which overlap the most unique region of each gene .
the microarray hybridization signal detected on these oligonucleotide elements revealed that these two highly homologous genes undergo different transcription regulation during the idc with h <dig> exhibiting 3-fold increase of mrna abundance in schizonts compare to trophozoites and h <dig>  showing similar amounts  between these two developmental stages .
taken together these data demonstrate that the newly designed microarray for p. falciparum successfully recapitulates data from previous transcriptome analyses and has a potential to further expand on these results.
overall these data verify the improved performance of oligorankpick in designing unique microarray elements for gene expression microarrays
discussion
the main goal of this work was to develop a microarray design algorithm which combines the thoroughness of the parameter optimization methods  and performs with high computational efficiency of the earlier, cutoff based techniques .
the newly developed algorithm, oligorankpick, is the first method using a parameter optimization approach that is computationally fast and robust for genome-wide microarray design.
the core principle of this technique consists of the rank transformations of the parameter scores and the subsequent weighted rank-sum strategy.
this allowed us to eliminate all cutoff based filters that are typically applied to the input data  or to partial oligonucleotide lists that are generated prior or during the decision-making step .
instead the derived rank-based system maintains all the oligonucleotide candidates in their rank order throughout the entire process.
this approach removes any ambiguities in the selection process as all oligonucleotides are constantly prioritized based on their properties.
since no oligonucleotides are eliminated by arbitrary cutoffs, this method also significantly expands the genome coverage of the designed microarrays.
the simplicity of the rank-based approach also allows the algorithm to perform gene specific optimizations of the weight coefficients in which the contribution of each parameter is modified based on the sequence properties of a particular gene.
this is especially useful for optimal probe design in genes with extreme parameters distributions such as high at content or high sequence homology to other genomic locus .
for example at richness of some genes causes the gc content parameter to be over emphasized due to a stronger priority that is given to the gc rich oligonucleotide windows.
this could force a selection of less unique oligonucleotides or oligonucleotides with complex secondary structure from these cg rich oligonucleotide candidates.
the implementation of the gene specific optimizations is likely the most innovative approach introduced by this method because it generates a tighter distribution for each oligonucleotide parameter compared to other publicly available programs .
for general functionality we derived and validate optimal weight set intervals which could be applied to a wide range of genomes.
the flexibility of the oligorankpick package, however, allows the users to tune these setting for other specialized applications.
for the development and validation of oligorankpick we design a new dna microarray for the most lethal species of the human malaria parasites p. falciparum whose genome was completed in  <dig>  <cit> .
we chose this genome for its extreme at/gc distribution and high level of gene duplication do demonstrate the utility of the newly design program for its future applications.
the average gc content in the p. falciparum genome is estimated  <dig> % .
for this design, however, we wished to select oligonucleotides with higher gc content to ensure higher tm and thus specificity and selectivity of each probe.
in addition the requirement for high gc content will help to select oligonucleotides with high sequence complexity as at rich sequences in p. falciparum contain numerous short nucleotide repeats.
as demonstrated in figure  <dig> oligorankpick was able to design a set of oligonucleotides whose gc content is tightly distributed around  <dig> %.
at the same time high levels of uniqueness and sequence complexity and a low level of secondary structures were preserved in the vast majority of the probes.
this feature of oligorankpick will be particularly useful for microarray design of many organisms with extreme fluctuations in gc content such as mycoplasma mycoides  <cit>  and other bacterial species  <cit> , other "at rich" plasmodium spp.
<cit>  and dictyostelium discoideum  <cit>  or gc rich leishmania spp.
<cit> .
the p. falciparum genome was found to contain a large number of duplicated genes sharing high levels of homology  <cit> .
the extreme examples are the three gene families  which are involved in the parasite virulence and are presently explored as potential molecular targets for malaria intervention strategies  <cit> .
despite the high levels of homology amongst the individual members of these gene families, oligorankpick was capable of designing specific oligonucleotide representative for  <dig> % of these genes which by far exceeded the performance of the three tested publicly available programs.
this improved performance will render oligorankpick useful for studies of many organisms with highly homologous, biologically significant gene families ranging from microbial pathogens  <cit>  to high eukaryotes  <cit> .
CONCLUSIONS
oligorankpick provides a powerful alternative for long oligonucleotide microarray design for genomes with extreme gc content fluctuations and high abundance of highly homologous gene families.
in its simplest implementation a user needs only to define the probe length and an expected gc content or tm.
however, for specialized applications, oligorankpick provides the user with the option of setting the range of relative importance  of each parameter as well as optimization of the quality control target values.
using this method we have designed and assembled a next generation of long oligonucleotide dna microarray for the main parasitic species of human malaria p. falciparum.
transcriptome analyses of two p. falciparum developmental stages demonstrated that the designed microarray provides the most comprehensive coverage of the p. falciparum genome presently available.
the oligonucleotide sequences and the transcriptome data are available from the supplementary file.

 dna microarray is one of the most powerful and versatile tools for post-genomic research. after the initial success with cdna and pcr product-based microarrays, application of long oligonucleotides became widely used in "spotted" dna microarray technology in the last five years. from the beginning it became clear that the design of the oligonucleotide probes requires special attention. under a single stringency condition, hybridization specificity and efficiency of all oligonucleotides must be globally maximized across the entire array. thus for the selection of the optimal oligonucleotide candidates, four major parameters are being evaluated: uniqueness which analyzes other possible cross-hybridization targets in the genome; sequence complexity which evaluates the presence of short nucleotide repeats; melting temperature or gc content which ensures a uniform hybridization efficiency across the microarray; and level of internal secondary structures which helps to avoid all possible self-binding interference with the specific target hybridization. in principle each of these properties can be calculated individually for every potential oligonucleotide candidate, however, the main challenge that remains is to derive a selection strategy that combines these parameters and selects the most optimal oligonucleotide representative for a given genetic locus/gene. all currently available programs for long oligonucleotide microarray design utilize different parameters: the binding energy or blast-based score to alternative targets to evaluate uniqueness, the gc content or tm to estimate hybridization stringency, the reverse smith-waterman score or free energy to evaluate levels of secondary structure and various types of complexity coefficients to evaluate the presence of short nucleotide repeats in each oligonucleotide element. typically these programs select one or more oligonucleotide representatives of a gene using various systems of cutoff-based filters. for example arrayoligoselector creates an intersection of oligonucleotides that pass parameter-based cutoffs for uniqueness, self-binding and sequence complexity. the intersection candidate list is then passed on to the gc filter and subsequently the final representative are selected using a 3' proximity criteria. the cutoff based algorithms provide a powerful approach to select dna microarray oligonucleotide sets and were successfully used to design dna microarrays for a large number of species. the use of these algorithms is, however, not completely optimal for genomes with high abundance of repetitive sequences and large fluctuations of gc content. to accommodate such genomic sequences, the methods must relax the parameter filter adjustments. the wide "opening" of the cutoff filters can cause selection of suboptimal oligonucleotides for a significant number of genes, due to the fact that all oligonucleotides that pass a particular filter are treated as equal by the subsequent steps, disregarding their subtle diversity within the filtered interval of the parameter. to overcome these shortcomings new algorithms which incorporate optimization strategies of oligonucleotide parameters were developed including oligodesign and commoligo. oligodesign was developed specifically for the design of the locked nucleic acid microarray platform which takes advantage of the improved nucleic on-chip capture sensitivity of the lna substitute mixmer oligonucleotides. design of these specialized probes requires careful optimizations of the hybridization specificity and efficiency for each probe. for this purpose, oligodesign uses an extensive fuzzification process derived from neural network approaches to ensure the optimal performance of this highly specialized microarray platform. similar to the fuzzy logic approach, commoligo uses a piece-wise linear function to select optimal oligonucleotides via a user configurable iterative process. both of these methods represented a step in the right direction, recognizing the need for parallel optimization of all used parameters and elimination of cutoffs that cause information loss. at its presently available implementation, however, both oligodesign and commoligo utilize complex and computer-time consuming processes that render them unsuitable for high throughput applications. nevertheless both methods have been useful for design of focused "miniarrays" which typically contain smaller numbers of genes e.g. stress response and toxicological markers from caenorhabditis elegans or microarrays for relatively small genomes such as methanoccocus maripaludis with genes. here we present a novel program named oligorankpick that is inspired by the aforementioned parameter optimization approaches and it is suitable for the design of gene specific long oligonucleotide probes for genomes of all sizes. the final decision making process is based on a weighted rank-sum strategy which significantly streamlines the entire computation process. this complete eliminates all cutoff based filters, thereby significantly improves the quality of the resulting microarray oligonucleotide design. moreover, the weighted rank-sum approach enables us to implement an integer weighted linear function to automatically optimize the oligonucleotide parameters for each gene individually. finally to demonstrate the utility of oligorankpick we design, assemble and verify a new version of p. falciparum microarray which comprises of oligonucleotides representing genes.  algorithm overview rank transformation several previous methods have been introduced to normalize oligonucleotide parameter scores including a piece-wise, and sigmoid function. in our approach the transformations of score values calculated for each parameter into rank numbers allows us to uniformly assesses and adjust the contribution of different parameters for the optimal oligonucleotide selection. for all coding sequences of the test genome there is a complete agreement between the overall profiles of the scores and the converted ranks in each parameter. this indicates that the rank transformation does not affect the oligonucleotide status in the oligonucleotide set, and that the rank transformation has an equal power of parameter comparisons to the original scores. optimizations of weight sets the challenge of deriving weight values that will select an optimal oligonucleotide in the rank-sum strategy is two-fold: the weight coefficients should correspond to the relative contribution of each parameter to the oligonucleotide performance during the microarray hybridization; the weight set optimization should provide sufficient flexibility to accommodate the variable nature of the primary structure along the genome. for this purpose we aimed to develop a strategy that optimizes the weight set for each gene individually by considering broader intervals of weight coefficients rather then a single target value. to derive and subsequently evaluate this strategy, we used the p. falciparum genome which is characterized by large fluctuations of gc content, and an abundance of repetitive sequences and large highly homologous gene families. in the first step we calculated the top oligonucleotide for all plasmodium genes using different weight sets. these sets originate from all combinations of four weight intervals: wblast=, wgc=, wsw=, wlz=. the broader intervals for uniqueness and cg content weight coefficients are intended to allow higher impact of these parameters on the final oligonucleotide selection. these adjustments are based on our previous observations, indicating that variations in gc content and uniqueness have a greater effect on specificity and efficiency of microarray hybridization compared to the other parameters and bozdech et al unpublished data). the implementation of weight interval optimization strategy then facilitates the gene specific optimizations of the oligonucleotide selection with respect to the sequence properties of a particular gene. previous studies suggest that 40% identity of continuous match is an upper limit for possible microarray cross-hybridization signal for oligonucleotides between and nucleotides. for the gc content a 5% deviation will result in a fluctuation in tm of 4°c which provides a theoretical upper limit for microarray hybridization stringency. we utilized these two rules ) as criteria to evaluate the quality of oligonucleotide selection. it is important to emphasize that these criteria are not implemented as cutoff values in the oligorankpick algorithm but as quality monitoring criteria to evaluate the selection process utilizing varying weight sets. first oligonucleotide sets of the p. falciparum genome were generated using all combinations of the initial weight values. then the number of oligonucleotides that passed the both rules was calculated. in the uniqueness weight simulations the number of oligonucleotides outside of the limit criteria gradually plateaus for the uniqueness weights greater than for wblast values greater then there were essentially no further oligonucleotide attritions regardless of the other weight values. by manual inspection we observed that the non-unique oligonucleotides originate mostly from duplicated genes for which no unique oligonucleotides could be selected. the gc content modeling exhibits essentially identical tendencies with the vast majority of oligonucleotides that were found outside of the gc range originating from genes with an extremely high at content. taken together both gc content and uniqueness weight values bellow do not generate enough power to maximize these parameters while values greater then do not improve the numbers of acceptable oligonucleotides due to the natural properties of the genome. thus in order to streamline the oligonucleotide design for the following studies, we built a limited weight pool composed of uniqueness weights and gc content from to sw weight is or and lz is defined as this scheme creates new optimized sets which can be used effective oligonucleotide selection. based on these simulations, we expect that the weight sets will provide sufficient flexibility and efficiency to optimize all four parameters, while preserving the higher importance of the gc content and uniqueness. in summary the oligorankpick algorithm is capable of designing microarray oligonucleotide sets avoiding any arbitrary cutoff implemented directly in the selection algorithm. instead this algorithm incorporates various empirical and theoretical boundaries via target values to optimize the simulation step during which specific weight intervals are determined. comparison with other programs to compare the performance of oligorankpick with other publicly available programs, we designed three theoretical microarray oligonucleotide sets for the p. falciparum, s. cerevisiae and e. coli. we selected three programs, arrayoligoselector, oligopicker and oligoarray. for the intended designs we chose the oligonucleotide length to be nt and the gc content % for p. falciparum, 40% for s. cerevisiae and 45% for e. coli. the theoretical oligonucleotide sets were designed using the publicly available sequence data and the selection algorithms with default settings. figure summarizes the parameter distributions of the uniqueness scores plotted against gc content. overall these contour plots illustrate that comparing to the three publicly available programs, oligorankpick provides significant improvements for the design of yeast, e. coli and p. falciparum microarray. the most striking improvements were, however, observed in the design of the p. falciparum microarray. for this genome the blast scores and the gc content of the oligonucleotides designed by oligorankpick exhibit a greater convergence to a small region in the desired area compared to oligonucleotides designed by the three other programs. similar convergence is observed for the sw and lz scores. to further demonstrate the convergence of the oligonucleotide parameters we calculated a mean distance for each parameter distribution to its desired value and also to the average value within the parameter distribution. in all cases the oligorankpick produced the smallest mean distances and thus tighter distribution of the oligonucleotide parameters. the only exception is the lower mean distance of the cg content from its mean value in the yeast set designed by oligopicker. detailed inspection of these results indicated that the low mean distance is due to extensive filtering implemented by this program. for each of the theoretical microarray dataset we also calculate the average weight score which is directly related to the oligonucleotide quality with respect to the oligonucleotide parameters. the smaller aws that are consistently observed for the oligorankpick generated oligonucleotide sets compared to the three other programs further indicate the optimization power of oligorankpick. *arrayoligoselector.4; oligopicker; oligoarray. a: oligonucleotide number selected by the program; b: good oligonucleotide number based on blast score of non-target and gc content. & percentage of good quality oligonucleotide to total selected oligonucleotide. ξ five rejected coding sequences are less than bp. ζ only one rejected sequence is yjr151w-a. one of the unique features of the p. falciparum genome is the presence of several large highly homologous gene families whose role has been implicated in the antigenic variation including var, rifin and stevor. table indicates the number of unique oligonucleotides designed by all the four programs for these genes. oligorankpick was capable of designing unique oligonucleotides for genes of total genes which by far exceeded the performance of the three other algorithms. design of a gene specific dna microarray for p. falciparum *arrayoligoselector.4; oligopicker; oligoarray. a: oligonucleotide number selected by the program; b: accepted oligonucleotide number based on blast score of non-target. in the final step we applied oligorankpick to design a gene specific dna microarray for the p. falciparum genome that can be used for functional genomic studies of this important human pathogen. for this design we wished to increase the oligonucleotide coverage for longer open reading frames and thus we fragmented each coding sequence using the fragmentation.pl script as follows: sequences smaller than kb were kept as one fragment; sequences between kb and kb were split evenly into two fragments, sequences larger then kb were split into n fragments when: kb < gene size > kb. the fragmentation step generated microarray element fragments from cds. a single oligonucleotide was designed for each mef which resulted in one oligonucleotide per bp on average for all p. falciparum coding sequences. although the median gc content of all nt oligonucleotide windows in the p. falciparum coding sequences is % for higher specificity and efficiency of microarray hybridization, we selected oligonucleotides with a gc content of %. oligorankpick successfully designed oligonucleotides representing all predicted p. falciparum genes with an average of oligonucleotides per protein coding sequence. figure 4b summarizes the gc content distribution suggesting that oligorankpick can identify optimal oligonucleotide elements with gc content significantly distant from the average gc content in the genome. astonishingly % of the designed oligonucleotides had the desired gc content of %. to evaluate the level of uniqueness of the designed oligonucleotides we used the identical quality control criteria used for the weight optimization strategy which is consistent with previously established conditions of optimal microarray hybridization performance. in total oligonucleotides passed the uniqueness criteria and oligonucleotides were found to be in the range of 5% deviation from the gc content target value. there are oligonucleotides meeting both criteria while only oligonucleotides were outside of the ± 5% gc content interval and oligonucleotides were not unique in the genome. manual inspections of the mefs represented by these oligonucleotides indicated that no suitable nt window exists within these dna fragments. the non-unique oligonucleotides represented genes from which genes belong to the large multigenic gene families, var,rifin and stevor. pair-wise sequence homology analysis of these genes revealed that these genes do not contain any nt window that shares less than 40% homology with any other member of the corresponding gene family and thus no unique oligonucleotide could be selected by any conceivable strategy. interestingly for the remaining members of these families a specific oligonucleotide was selected which further demonstrates the power of oligorankpick for microarray design. transcriptome analysis of the trophozoite and schizont stages of p. falciparum although all parameters of the oligonucleotide microarray sets designed by oligorankpick indicate their high quality, the ultimate evidence for their functionality can be provided only by physical microarray experiments. for this purpose we have synthesized all the oligonucleotides for the p. falciparum genome-wide microarray and spotted these onto polylysine-coated microscopic slides as previously described. using these microarrays we compare the global mrna patterns between two developmental stages of the p. falciparum intraerythrocytic development, trophozoite and schizont. all experimental procedures were carried out as previously described and the complete results for three replicates of the microarray hybridizations are available in the supplementary data. the p. falciparum genome sequence reference strain 3d was used for this analysis. total genes were found to be expressed in at least one of the studied developmental stages in three replicates of microarray hybridization. from these and mrna transcripts exhibited at least 2-fold higher abundance in the trophozoite and the schizont stage, respectively. in order to assess the fidelity of the obtained results we wish to compare this data to previously published transcriptome analyses of the p. falciparum intraerythrocytic developmental cycle. these include the idc transcriptome analyzed by the previous version of a long oligonulceotide microarray comprised of genes, and a high density short oligonucleotide affymetrix microarray dataset comprised of genes with stage specific transcription. all genes present in both lom-idc and hdso-affymetrix transcriptomes were represented on the new p. falciparum microarray and yielded a hybridization signal in at least two of the three microarray replicates. to compare the stage specificity of the gene expression we select genes which exhibited > 3-fold change in mrna abundance between trophozoite and schizonts detected in at least two replicates. using these criteria we classify genes as trophozoite specific and genes as schizont specific. the transcriptome data comparisons, summarized in table indicate high correlations between the transcriptome data and the new microarray dataset with – % of overlapping genes exhibiting identical stage specificity in their mrna levels. there were only a small number of genes for which the new expression results did not correlate with the previously published data. these discrepancies are likely caused by subtle differences in parasite culture synchronicity and stage representation between our culturing system and the systems used for the previous transcriptome analyses. *genes with peak expression before and after hours post invasion are classified as trophozoite and schizont specific, respectively ** genes with higher expression levels in late ring and early and late trophozoites compared to early and late schizonts are classified as trophozoite specific and vice versa to further validate the performance of the designed p. falciparum microarray quantitative real-time rt-pcr was used to measure relative mrna abundance between trophozoite and schizont stage for selected genes. for this we chose genes for which only oligorankpick designed a "good quality" microarray element while the three tested publicly available programs did not yield a suitable oligonucleotide element. these include two paralogous histone five members of the variable surface antigen gene families, centrin, and two genes encoding highly homologous hypothetical proteins. figure 5a shows good correlations between the rt-pcr results and microarray hybridization data which demonstrate the robust performance of the newly designed microarray for analyses of mrna abundance in p. falciparum. detail sequence analyses revealed that each of the selected genes contains only a small window of unique sequence while the majority of the gene is highly homologous to at least one other locus in the genome. one of the example is a pair of highly homologous genes encoding histone and its homologue histone. this high homology is likely the main obstacle for designing a specific oligonucleotide and it is the reason why no transcription data have been obtained by the previously reported transcriptome analyses. despite this oligorankpick selected specific oligonucleotides which overlap the most unique region of each gene. the microarray hybridization signal detected on these oligonucleotide elements revealed that these two highly homologous genes undergo different transcription regulation during the idc with h exhibiting 3-fold increase of mrna abundance in schizonts compare to trophozoites and h showing similar amounts between these two developmental stages. taken together these data demonstrate that the newly designed microarray for p. falciparum successfully recapitulates data from previous transcriptome analyses and has a potential to further expand on these results. overall these data verify the improved performance of oligorankpick in designing unique microarray elements for gene expression microarrays discussion the main goal of this work was to develop a microarray design algorithm which combines the thoroughness of the parameter optimization methods and performs with high computational efficiency of the earlier, cutoff based techniques. the newly developed algorithm, oligorankpick, is the first method using a parameter optimization approach that is computationally fast and robust for genome-wide microarray design. the core principle of this technique consists of the rank transformations of the parameter scores and the subsequent weighted rank-sum strategy. this allowed us to eliminate all cutoff based filters that are typically applied to the input data or to partial oligonucleotide lists that are generated prior or during the decision-making step. instead the derived rank-based system maintains all the oligonucleotide candidates in their rank order throughout the entire process. this approach removes any ambiguities in the selection process as all oligonucleotides are constantly prioritized based on their properties. since no oligonucleotides are eliminated by arbitrary cutoffs, this method also significantly expands the genome coverage of the designed microarrays. the simplicity of the rank-based approach also allows the algorithm to perform gene specific optimizations of the weight coefficients in which the contribution of each parameter is modified based on the sequence properties of a particular gene. this is especially useful for optimal probe design in genes with extreme parameters distributions such as high at content or high sequence homology to other genomic locus. for example at richness of some genes causes the gc content parameter to be over emphasized due to a stronger priority that is given to the gc rich oligonucleotide windows. this could force a selection of less unique oligonucleotides or oligonucleotides with complex secondary structure from these cg rich oligonucleotide candidates. the implementation of the gene specific optimizations is likely the most innovative approach introduced by this method because it generates a tighter distribution for each oligonucleotide parameter compared to other publicly available programs. for general functionality we derived and validate optimal weight set intervals which could be applied to a wide range of genomes. the flexibility of the oligorankpick package, however, allows the users to tune these setting for other specialized applications. for the development and validation of oligorankpick we design a new dna microarray for the most lethal species of the human malaria parasites p. falciparum whose genome was completed in. we chose this genome for its extreme at/gc distribution and high level of gene duplication do demonstrate the utility of the newly design program for its future applications. the average gc content in the p. falciparum genome is estimated %. for this design, however, we wished to select oligonucleotides with higher gc content to ensure higher tm and thus specificity and selectivity of each probe. in addition the requirement for high gc content will help to select oligonucleotides with high sequence complexity as at rich sequences in p. falciparum contain numerous short nucleotide repeats. as demonstrated in figure oligorankpick was able to design a set of oligonucleotides whose gc content is tightly distributed around %. at the same time high levels of uniqueness and sequence complexity and a low level of secondary structures were preserved in the vast majority of the probes. this feature of oligorankpick will be particularly useful for microarray design of many organisms with extreme fluctuations in gc content such as mycoplasma mycoides and other bacterial species, other "at rich" plasmodium spp. and dictyostelium discoideum or gc rich leishmania spp.. the p. falciparum genome was found to contain a large number of duplicated genes sharing high levels of homology. the extreme examples are the three gene families which are involved in the parasite virulence and are presently explored as potential molecular targets for malaria intervention strategies. despite the high levels of homology amongst the individual members of these gene families, oligorankpick was capable of designing specific oligonucleotide representative for % of these genes which by far exceeded the performance of the three tested publicly available programs. this improved performance will render oligorankpick useful for studies of many organisms with highly homologous, biologically significant gene families ranging from microbial pathogens to high eukaryotes.  oligorankpick provides a powerful alternative for long oligonucleotide microarray design for genomes with extreme gc content fluctuations and high abundance of highly homologous gene families. in its simplest implementation a user needs only to define the probe length and an expected gc content or tm. however, for specialized applications, oligorankpick provides the user with the option of setting the range of relative importance of each parameter as well as optimization of the quality control target values. using this method we have designed and assembled a next generation of long oligonucleotide dna microarray for the main parasitic species of human malaria p. falciparum. transcriptome analyses of two p. falciparum developmental stages demonstrated that the designed microarray provides the most comprehensive coverage of the p. falciparum genome presently available. the oligonucleotide sequences and the transcriptome data are available from the supplementary file. 
in comparison to other published algorithms oligorankpick provides significant improvements in oligonucleotide design for all three genomes with the most significant improvements observed in the microarray design for p. falciparum whose genome is characterized by large fluctuations of gc content, and abundant gene duplications. currently, all of the publicly available programs for dna long oligonucleotide microarray selection utilize various combinations of cutoffs in which each parameter is evaluated and filtered individually. the use of the cutoffs can, however, lead to information loss and to selection of suboptimal oligonucleotides, especially for genomes with extreme distribution of the gc content, a large proportion of repetitive sequences or the presence of large gene families with highly homologous members. the weighted rank-sum strategy utilized by this algorithm provides high flexibility of oligonucleotide selection which accommodates extreme variability of dna sequence properties along genomes of many organisms. the design of long oligonucleotides for spotted dna microarrays requires detailed attention to ensure their optimal performance in the hybridization process. the main challenge is to select an optimal oligonucleotide element that represents each genetic locus/gene in the genome and is unique, devoid of internal structures and repetitive sequences and its tm is uniform with all other elements on the microarray. here we present the program oligorankpick which is using a weighted rank-based strategy to select microarray oligonucleotide elements via an integer weighted linear function.
 the design of long oligonucleotides for spotted dna microarrays requires detailed attention to ensure their optimal performance in the hybridization process. the main challenge is to select an optimal oligonucleotide element that represents each genetic locus/gene in the genome and is unique, devoid of internal structures and repetitive sequences and its tm is uniform with all other elements on the microarray. currently, all of the publicly available programs for dna long oligonucleotide microarray selection utilize various combinations of cutoffs in which each parameter is evaluated and filtered individually. the use of the cutoffs can, however, lead to information loss and to selection of suboptimal oligonucleotides, especially for genomes with extreme distribution of the gc content, a large proportion of repetitive sequences or the presence of large gene families with highly homologous members.  here we present the program oligorankpick which is using a weighted rank-based strategy to select microarray oligonucleotide elements via an integer weighted linear function. this approach optimizes the selection criteria for each gene individually, accommodating variable properties of the dna sequence along the genome. the designed algorithm was tested using three microbial genomes escherichia coli, saccharomyces cerevisiae and the human malaria parasite species plasmodium falciparum. in comparison to other published algorithms oligorankpick provides significant improvements in oligonucleotide design for all three genomes with the most significant improvements observed in the microarray design for p. falciparum whose genome is characterized by large fluctuations of gc content, and abundant gene duplications.  oligorankpick is an efficient tool for the design of long oligonucleotide dna microarrays which does not rely on direct oligonucleotide exclusion by parameter cutoffs but instead optimizes all parameters in context of each other. the weighted rank-sum strategy utilized by this algorithm provides high flexibility of oligonucleotide selection which accommodates extreme variability of dna sequence properties along genomes of many organisms. 