 Methodology
 Open Access
 Published:
A multiobjective gene clustering algorithm guided by apriori biological knowledge with intensification and diversification strategies
BioData Mining volume 11, Article number: 16 (2018)
Abstract
Background
Biologists aim to understand the genetic background of diseases, metabolic disorders or any other genetic condition. Microarrays are one of the main highthroughput technologies for collecting information about the behaviour of genetic information on different conditions. In order to analyse this data, clustering arises as one of the main techniques used, and it aims at finding groups of genes that have some criterion in common, like similar expression profile. However, the problem of finding groups is normally multi dimensional, making necessary to approach the clustering as a multiobjective problem where various cluster validity indexes are simultaneously optimised. They are usually based on criteria like compactness and separation, which may not be sufficient since they can not guarantee the generation of clusters that have both similar expression patterns and biological coherence.
Method
We propose a MultiObjective Clustering algorithm Guided by aPriori Biological Knowledge (MOCGaPBK) to find clusters of genes with high levels of coexpression, biological coherence, and also good compactness and separation. Cluster quality indexes are used to optimise simultaneously gene relationships at expression level and biological functionality. Our proposal also includes intensification and diversification strategies to improve the search process.
Results
The effectiveness of the proposed algorithm is demonstrated on four publicly available datasets. Comparative studies of the use of different objective functions and other widely used microarray clustering techniques are reported. Statistical, visual and biological significance tests are carried out to show the superiority of the proposed algorithm.
Conclusions
Integrating apriori biological knowledge into a multiobjective approach and using intensification and diversification strategies allow the proposed algorithm to find solutions with higher quality than other microarray clustering techniques available in the literature in terms of coexpression, biological coherence, compactness and separation.
Background
During the last two decades, the explosion in the increase of DNA microarray datasets available has promoted the application of machine learning methods for the understanding of the genomic data. A DNA microarray is used to collect information regarding gene expression level [1] under different conditions like a time series during a biological process, experiments of different tissue samples, among others [2]. This highthroughput technology has allowed a fast progress in biological and biomedical research [3], and it has facilitated the study of problems such as differential gene expression [4, 5], patterns of genes with (dis)similar expression levels [6–8], prediction of response to treatment [9, 10] and detection of gene mutations [11].
Clustering has proved to be a useful unsupervised learning technique for gene expression data analysis [12]. The goal is to find a partition of genetics elements represented in the microarray into k distinct groups, where k is the number of clusters which may or may not be known in advance. It is expected that genetic elements such as gene, EST contigs, noncoding sequences, among others, with similar expression profiles be put into a single cluster [13] as a way to reveal hidden patterns. Carrying out clustering is not a trivial task, in fact this unsupervised learning technique currently remains a complex and challenging task which was proved to be an NPhard problem [14]. Clustering can also be seen as an optimisation problem [15] where a cluster index (objective function) is optimised to obtain clustering solutions of high quality.
Several clustering algorithms for gene expression data have been proposed during the last years [16–24]. They are based on guiltbyassociation paradigm [25], i.e. groups of genetics elements which are associated, share similar expression profiles are more likely to share function. In recent years, this paradigm has been reformulated because the optimisation of a single cluster quality index based on expression levels can cause some issues. The fact that two genetic elements have similar expression patterns can be because they share some functionality, but also because of noise, which may lead to the misidentification of biological relationships [26]. Because of the above, some authors used external biological knowledge [12, 27] as another source of information about genetics elements as a way to address this situation and to find clusters with more biological coherence. Often external biological knowledge concerns the use of repositories such as the Gene Ontology Project (GO) [28] or Kyoto Encyclopedia of Genes and Genomes (KEGG) [29]. However, this biological knowledge can be partial, i.e. the information could be available only for a subset of the genetic elements. Clearly, using only a quality index based on external biological knowledge will lead to a partition of the data with clusters previously discovered and thereby extract repetitive information.
Clustering methods can use the expression profilesbased distance (D_{EB}) or biologicalbased distance (D_{BB}) to cluster genes. The distance value represents the strength of the relationship between two genes, in terms of expression behaviour (D_{EB}) or biological functions (D_{BB}). The distance (D_{EB} and D_{BB}) between two clusters is calculated as the distance between the medoids (the most central gene located in a cluster) of each cluster. This process helps to uncover new relationships in terms of cellular functions and biological processes in which genes participate; as well as, to understand their interactions, and cellular regulation. These results can also aid the study of the influence of genes in the development of diseases, their association in the formation of tissues or groups of genes that have similar response to a given drug.
The clustering problem can be naturally address as a mutiobjective problem [30], where we want to improve objectives related to expression similarity and biological coherence (one or more sources). The multiobjective clustering (MOC) problem can be described as:
where Ω is the set of all feasible clustering solutions, C is a clustering solution, and P_{t},t=1,…,m is a set of m different objective functions (quality indexes), i.e. that clustering C^{∗}⊆Ω corresponds to clustering solutions that have the best optimised m criteria P [30, 31].
In MOC problems, we have several clustering solutions C^{∗} that correspond to the optimisation of objective functions that tend to be in conflict [32], i.e. improving one objective involves worsening another. Then, it is required to reach a “tradeoff” where all objective functions (quality indexes) are satisfied to an acceptable degree which leads to find a set of best solutions called nondominated solutions [30].
Nondominated solutions Given two clustering solutions C_{1} and C_{2}∈Ω, solution C_{1} is said to dominate solution C_{2} (denoted as C_{1}≺C_{2}) (minimisation) if and only if:
Pareto optimal set Pareto optimal set Π is defined as:
Thus, a Pareto optimal set Π corresponds to a set of nondominated solutions, such that there is no other solution in Ω that dominates any of them.
Pareto front The Pareto Front F^{∗} corresponds to the image of Pareto optimal set Π, i.e. to the vectors of criterion functions (quality indexes) to Π.
In the literature it is possible to find multiobjective clustering (MOC) approaches for clustering expression data. The work presented in [33, 34] used a multiobjective genetic algorithm along with supervised techniques to perform the clustering process optimising two cluster quality indexes based on gene expression levels. However, biological information about genes is only used as verification and it is not part of the process of generating clusters. In [35] authors propose a technique for clustering of genes biologically guided by the interaction of a decision maker (DM). The technique optimises several cluster quality indexes based on expression level, meanwhile the DM evaluates clustering solutions based on the relationship in biological terms according to the expert knowledge in the area. Although the approach is interesting, since it considers expression and biology information, the fact that the formation of clusters is affected by DM expertise makes the approach limited to experiments with data in a small area where the decision maker has experience. In these multiobjective clustering approaches for gene expression data it is observed that only few of them use biological knowledge in spite of works in [26, 36, 37], which have shown that the inclusion of biological knowledge during the clustering process allows to find gene clusters with more common biological properties. In this paper, we present a multiobjective clustering algorithm guided by apriori biological knowledge. The proposed algorithm is based on NonDominated Sorting Genetic Algorithm (NSGAII) [38] which includes intensification and diversification strategies based on both PathRelinking (PR) [39] and Pareto Local Search (PLS) [40], respectively. The main contributions of this work are (1) the integration of information of genetic elements regarding their levels of expression and biological functions during the optimisation of cluster quality indexes, and (2) the proposal of adhoc local search strategies that exploit both the memory mechanism and neighbourhood principles of PathRelinking (PR) and Pareto Local Search (PLS) respectively, using a multiobjective approach. This work is tested against state of the art algorithms in order to show the benefits of both: using a multiobjective approach to tackle the clustering of expression data and the method proposed is able to discover clusters with more stronger corelation and common biological properties than literature alternatives.
Method
We call our method “MultiObjective Clustering Guided by aPriori Biological Knowledge” (MOCGaPBK). The method uses biological knowledge by the means of annotations of genetic elements with Gene Ontology (GO) terms. The integration of this biological knowledge is performed by the computation of the biologybased (D_{BB}) and the expressionbased (D_{EB}) distances.
Biologybased distance (D_{BB}) We use the Wang functional similarity (WS) [41]. It is an information content (IC) based metric which determines the similarity of two Gene Ontology (GO) terms based on both the location of these terms in the GO graph and their relation with their ancestor term. Given two elements G_{x} and G_{y} annotated by GO term sets GO_{1}={go_{11},go_{12},…,go_{1m}} and GO_{2}={go_{21},go_{22},…,go_{2m}} respectively, their WS is represented as WS(G_{x},G_{y}) with values that can vary between 0 to 1. For more detail on the computation of Wang similarity refer to [41]. We transform the Wang similarity into a distance measure using function 4.
Expressionbased distance (D_{EB}) We use Pearson correlation coefficient (ρ). It is actually a measure of similarity indicating how and how strongly level expression of two elements (Gx, Gy), for m different conditions, are related. We compute:
In Fig. 1 we show the incorporation process of the biological knowledge. First, we compute the distance matrices D_{EB} and D_{BB}. Then, each objective function is computed for two cases: (1) using biologybased distance (D_{BB}) and (2) using expressionbased distance (D_{EB}). Thus, we will be simultaneously optimising expression level similarity and biological functionality to discover clusters with high levels of coexpression and biological coherence.
Multiobjective clustering process
Our approach performs the discovery of clusters using NSGAII algorithm [38] along with PathRelinking (PR) [42] and Pareto Local Search (PLS) [43] as intensification and diversification strategies, respectively.
Algorithm 1 shows the pseudocode of the MOCGaPBK algorithm. First, it computes the expression (D_{EB}) and biological distance (D_{BB}) matrices (lines 12) in order to integrate the biological knowledge. Then the algorithm creates an initial population P_{0} of size N (line 4). In each generation v, the algorithm creates an offspring population Q_{v} of N individuals by using a binary tournament selection, the (k1)point crossover and the controllerrandom mutation operations (lines 67). A population R_{v} of size 2N (line 8) is created by union of P_{v} and Q_{v} (line 8). Then, a nondominated sorting and crowding distance calculation is applied on R_{v}. Here, solutions are ranked according to their nondomination level in F_{1},F_{2},…,F_{n}., i.e. NSGAII, label as F_{1} to nondominated solutions, F_{2} to nondominated solutions remaining after eliminating those with F_{1}. The process is repeated until it sorts all solutions in R_{v}. Then, in R_{v}, only those nondominated solutions, that is, the solutions ranked as F_{1}, are maintained (lines 910). The R_{v} population is used as input for the intensification and diversification strategies (lines 1114). The next population P_{v+1} is created by selecting the solutions labeled as F_{1} in R_{v} (line 20). If F_{1}<N, the algorithm complements the population with random solutions P_{r}, P_{v+1}=F_{1}∪P_{r} (lines 1618). On each generation the algorithm evaluates whether it reaches either of the two stopping criteria: (1) when the number of generations is reached and (2) when during a certain number of generations have passed without changes in the values of the objective functions of the solutions in the Pareto frontier. Finally, the algorithm returns the set of nondominated solutions (Pareto Front) in R_{v} (line 24).
Chromosome encoding
We use an integer encoding to represent a solution. Each number represents a cluster medoid which is the most central element located in a cluster, i.e. whose sum of the distances to another element of the cluster is minimum [44]. Each chromosome ch has the same length as the number of clusters K, and each position ch_{i} can have an integer value from 1 to n, n being the number of elements in the dataset. For instance, in case of a dataset with 100 elements, the chromosome [1 6 19 83 14 3] represents six clusters with 1, 6, 19, 83, 14, and 3 as the centre of each clusters.
Initial population
The initial population is generated as follows: first, the 50% of chromosomes is randomly created. Second, the remaining 50% is created based on a single genetic algorithm (SGA) which uses an integer encoding to represent a solution, k1 point crossover and controllerrandom mutation as evolutionary operators. Here, we optimise the PBM [45] index which is calculated using the distances between the points and their barycenters and the distances between the barycenters themselves. This initialisation helps the convergence because it gives to the multiobjective clustering algorithm good initial solutions.
Selection
We use a binary tournament selection along with Pareto ranking and crowding distance. In order to identify the winner chromosomes of each tournament, the individual belonging to the top Pareto ranking is chosen and in the case of a tie, the one with the highest crowding distance is selected.
Crossover and mutation
We use a (k1)point crossover, and a controllerrandom mutation [46] as evolutionary operators. In the crossover, k1 points on both individuals (parents) are randomly selected. All cluster medoid between those points are swapped between the two individuals. In controllerrandom mutation, a random position is selected from the chromosome and the corresponding element is replaced by a randomly chosen element that is not in the chromosome.
Objective functions
Three cluster validity indexes are selected as objective functions: XieBeni [47], Overall Cluster Deviation [48] and Cluster Separation [49]. They have been chosen because they are based on medoids and they have been used as objective functions in other multiobjective evolutionary clustering algorithms, since they are able to measure compactness and separation of the clusters which are the main properties evaluated in clustering task. They allow the multiobjective evolutionary clustering algorithms to be able to optimise simultaneously multiple characteristics of the data, while encouraging the formation of more homogeneous clusters and more higher separation between clusters at the same time. Here, we assumed notations as in [44]:

n: Number of elements in dataset.

K: Number of clusters.

x_{i}: ith element in dataset, with i=1,2,…,n

z_{k}: kth cluster medoid.

C: Set of all clusters.

C_{k}: kth cluster.
Table 1 shows a summary of the three cluster validity indexes used as objective functions.
Note that validity indices in Table 1, the distance D is computed using both expression profilesbased distance (D_{EB}) and biologicalbased distance (D_{BB}). Thus, each objective function has two versions: expressionbased (EB) and biologybased (BB) indices which consider Pearson correlation (ρ) and Wang functional similarity (WS) respectively. For instance, when XB index is used, we have two objective functions: XB _{EB} and XB _{BB}.
Intensification and diversification strategies
MOCGaPBK algorithm applies intensification and diversification strategies where promising regions are thoroughly explored. The strategies are based on Pathrelinking (PR) [42] and Pareto Local Search (PLS) [43], respectively.
Multiobjective pathrelinking
PR was originally proposed as an approach to integrate intensification strategy in the context of tabu search or scatter search. PR generates new solutions by exploring trajectories that connect highquality solutions. It starts from one of these solutions, called start solutions, and generates a trajectory in the neighbourhood space that leads toward the other solutions, called guiding solutions [50]. Our PR strategy is based on the implementation presented in [51, 52] but we adapted it to multiobjective clustering. Figure 2 shows a schematic representation for the construction of trajectories in the multiobjective pathrelinking (MOPR).
The algorithm builds trajectories between solutions as follows: Let C_{1} and C_{2} be two solutions obtained from the Pareto front (PF) of the multiobjective clustering process. The Pathrelinking procedure PR (C_{1},C_{2}) starts with the initial solution C_{1}, and gradually transforms it into solution C_{2}, by swapping out medoids in C_{1} and replacing them with medoids in C_{2}. To choose the initial solutions C_{1} and C_{2}, the algorithm selects the two solutions with the lowest Pareto ranking and the highest crowding distance. Then, for the next pair of solutions \(C^{\prime }_{1}\) and \(C^{\prime }_{2}\), we set \(C^{\prime }_{1} \leftarrow C_{2}\) as the current initial solution and it selects \(C^{\prime }_{2}\) as a new solution from the Pareto front with the lowest Pareto ranking and the highest crowding distance not considered before. This process continues until each solution in the Pareto front (PF) has been selected.
To gradually transform C_{1} into C_{2}, only nonrepeated medoids are considered as follows: Let \(M_{C_{1}}\) be the set of medoids in C_{1}. Let \(M_{C_{1}}M_{C_{2}}\) be the set of medoids in C_{1} not present in C_{2} and symmetrically, let \(M_{C_{2}}M_{C_{1}}\) be the set of medoids in C_{2} not present in C_{1}. Let tr_{0}(C_{1},C_{2})=C_{1} be the start solution in the trajectory TR(C_{1},C_{2}) from C_{1} to C_{2}. To obtain the next solution tr_{1}(C_{1},C_{2}) in the trajectory TR(C_{1},C_{2}), we remove from C_{1} a single medoid \(z_{k} \in M_{C_{1}}M_{C_{2}}\phantom {\dot {i}\!}\), and replace the empty position adding a medoid \(z_{l} \in M_{C_{2}}M_{C_{1}}\phantom {\dot {i}\!}\). To choose the solution in tr_{n} with the best move in each trajectory, we conducted a nondominated and crowding distance sorting. After that, we select the top ranked solution which is the new start solution C_{1}. The procedure is repeated until we reach the guiding solution C_{2}. Finally, each solution with the best move and original start and guiding solutions are stored in an set of intermediate pool (IP) solutions.
In our experiments, we apply PR(C_{1},C_{2}) and PR(C_{2},C_{1}) for each pair C_{1} and C_{2} in PF. Then, we merge IP and PF solutions and we recheck their nondominating and crowding distance levels. The nondominated solutions labelled as F_{1} are returned as the output of the procedure and it corresponds to the new Population R.
Pareto local search
To improve the Pareto solutions found by MOPR procedure, we implement a Pareto Local Search (PLS) [40] method based on the Pareto dominance criterion. PLS explores the Pareto neighbourhood of a set of nondominated solutions until it reaches a local optimal Pareto front [53]. A schematic representation of PLS is shown in Fig. 3.
Here, the PLS procedure receives a population of nondominated solutions A_{0} which are marked as unexplored. Then they are duplicated in a population A (Fig. 3a). An iterative process of three steps is performed. Selection step randomly selects a solution C from population A_{0}. After, neighbourhood exploration chooses a medoid z_{k} of C, which is then replaced in all neighbouring solutions N(C)={C1′,C2′,…,Cn′} by a medoid z_{k} that has not been assigned to the current solutions in N(C). Meanwhile, a dominating concept is used as acceptance criterion step,.i.e. the procedure will evaluate each C^{′}∈N(C) and if \(C \nprec C'\) it will mark as unexplored to C^{′} and the population A is updated eliminating those solutions are dominated by C^{′} (Fig. 3b). Once all C^{′}∈N(C) are evaluated, solution C is marked as explored and population A_{0} is updated filling only with the unexplored solutions from population A (Fig. 3c). PLS ends when the population A_{0} has no solutions, i.e., when all the solutions in population A have been explored, and they correspond to the new Population R.
Selection of a single solution
The MOCGaPBK algorithm produces a final Pareto front (PF) with a collection of one or more nondominated solutions. All of these solutions are highquality gene expression data partitions. To compare our results with those available in the literature, we select a single solution based on Silhouette index (S) [54] using the last nondominated solutions set in population R. The solution with the maximum value of S index is selected.
The Silhouette index quantifies the goodness of any clustering solution C measuring how similar an element is to its own cluster (compactness) compared to other groups (separation). It is calculated as follows:
This index score lies between − 1 and 1, so that values close to 1 indicates better clustering solutions. To more detail of S(C) equation refer to [44].
Results
All the algorithms used in the experiment were implemented using R [55] version 3.2.5 and computational tests performed on a computer with Intel Xeon E52650V4 30 MB, 4 CPUs, 2.2Ghz, 96 cores/threads, 128GB RAM, 4TB. The distance between expression profiles were calculated using functions of the “amap” library [56]. The distance of biological functionality using the “GOSemSim” library [57].
Datasets
Datasets used for experiments correspond to four reallife microarray gene expression datasets: arabidopsis thaliana [58], yeast cell cycle [59], yeast cell sporulation [60], and human fibroblasts serum [61] which were taken from here [62]. Here, duplicated elements and missing values of expression levels are removed. Expression levels are normalised so that each row has mean 0 and variance 1 (Table 2).
Experimental parameters
The algorithm is executed with the following parameters: number of generations =100, population size =50, crossover probability =0.80, mutation probability =0.01, generations without improvement =10, number of clusters k={4,5,6}. Evolutionary parameters and k values, were set up considering similar configurations used by other MOC algorithms in [34, 35, 63].
Performance evaluation
To evaluate the performance of MOCGaPBK algorithm, first we determine the best combination of objective functions regarding the hypervolume (HV) indicator. Then, we compare the performance of MOCGaPBK facing contestant clustering algorithms, under three aspects: (1) levels of coexpression, (2) biological coherence, and (3) compactness and separation. To carry out that, we use Eisen [64] and cluster profile [65] plots; annotation enrichment analysis [66]; and Silhouette index, respectively.
Hypervolume (HV) It is a unary metric that measures the volume (area in our case) in the objective function space covered by members of a Pareto set PS [67]. The hypervolume for every solution i∈PS computes an area a_{i} regarding a reference point W. Thus, the union of all areas a_{i} define the hypervolume value as follows:
Eisen plot It is a tool used in microarray experiment for visual representation of gene expression profiles. It is achieved using heat map and colouring values that usually are red, black and green [65]. Here, Eisen plot is used to show clustering results so that, similar colours are grouped together, showing that the expression profiles of the genes of a cluster are similar to each other.
Cluster profile plot This tool shows gene expression of microarray using x, y matrix representation of time points and level expression [65], respectively. Here, the normalised level expression values (in green) of genes in each cluster are used. Additionally, the average expression along with the standard deviation (in black) are included.
Annotation enrichment analysis It determines the biological relevance of a cluster regarding shared functions between those genes within it [68]. It requires functional annotations to obtain such information, standing out GO. It has three main terms: “biological process”, “molecular function”, and “cellular component”; containing biological information from a large list of genes. It uses a cumulative hyper geometric distribution to determine the degree of functional enrichment (pvalue) of overlap between annotations made to a given gene set. Thus, as shown in [34] for a particular GO term, the probability p of getting k or more genes within a cluster of size n, can be calculated as follows:
where f represents the total number of genes in a GO category and g the total number of genes within the genome. Thus, this test measures the degree of overlap between the genes in each group and the genes in GO category.
Experimental results
Effect of objective functions
To determine the effect of the objective functions during data clustering, we show the best Pareto front regarding hypervolume values obtained over 20 consecutive runs for each one in all datasets (Table 3).
To calculate the objective space covered by such objective functions, we use (1,1) as normalised reference point W. Note that higher hypervolume values implies better results from a multiobjective point of view. In Fig. 4, we show the best Pareto fronts for each objective functions. Here, further away nondominated solutions are from the reference point, a larger size of the solutions space will be covered and then better results will be achieved.
Effect of local search strategies
Similarly, to determine the effect of local search methods to improve the multiobjective gene clustering process, in Fig. 5, we compare the best Pareto front obtained by MOCGaPBK algorithm and three variations: using only NSGAII, NSGAII + PathRelinking (PR) and NSGAII + Pareto Local Search (PLS). To compute it, we consider the best combination (Table 3) of objective functions, i.e., XB _{EB} and XB _{BB} over 20 consecutive runs for all k values and datasets.
Eisen and cluster profile plots
The best hypervolume value is reached when the objective functions XB _{EB} and XB _{BB} are optimised. In Figs. 6, 7, 8 and 9, we show the Eisen and cluster profile plots of the solution with the best silhouette value found by MOCGaPBK. In Eisen plot, clusters are separated using a white line and genes are ordered according to the group to which they belong. In Eisen plots, we can see that each cluster has similar color patterns, denoting that expression profiles throughout the samples of the genes within each cluster are similar to each other. In the same way, the cluster profile plots show how the curve that represents the expression profiles of genes along the samples are similar within cluster. However, expression profiles inter clusters differ from each other. Hence, both plots show that our algorithm, with objective functions XB _{EB} and XB _{BB}, achieves superior performance to obtain coexpressed genes.
Annotation enrichment analysis
To demonstrate the biological coherence of clusters yield by MOCGaPBK algorithm, we use the FatiGO[69] web tool. It applies a functional enrichment test that evaluates the number of genes in each cluster annotated to a particular GO term. We consider the geneannotation tables based on the biological processGO term at 1% of significant level. Table 4 contains the three most significant GO terms along with their pvalues measured by the cumulative hypergeometric distribution.
Discussion
We evaluate the performance of MOCGaPBK algorithm using three combination of objective functions regarding hypervolume indicator. Table 3 provides information of the effect of them in clustering process which reveals the effectiveness of using XB _{EB}  XB _{BB} since it achieves the best values of hypervolume in all datasets. Such information can also be observed in Fig. 4, where the Pareto front of both XB criteria (cross symbol) dominates most of the solutions in the Pareto optimal set of the other objective functions, due to being further away from the reference point. In the same figure, the gray area represents the objective space covered only by the objective functions XB _{EB} and XB _{BB}. Such situations show that they are the best combination to perform the gene clustering process with our MOCGaPBK algorithm.
We also compare the performance of multiobjective optimization process regarding the incorporation of local search strategies. Figure 5 shows the best Pareto frontiers yield by MOCGaPBK and its variations on each of the four datasets. Generaly speaking, the use of local search improves the solutions found by the NSGAII, but it is not conclusive since for all datasets there are solutions found by NSGAII that dominates some of the solutions produced when a single local search strategy is used. However, the Pareto frontier produced by MOCGaPBK mostly dominates the Pareto frontiers produced by the other algorithms. In particular, for the case of the Yeast Cell Cycle data set (Fig. 5b), our proposal dominates all the solutions of the other algorithm, while in the other cases (Fig. 5a, c and d) there are only a few solutions that are non dominated. Also, MOCGaPBK always dominates all the solutions found by NSGAII. In all of the figures the gray area shows the space that is being covered by MOCGaPBK and not by the others. These results show the positive effect in the NSGAII of including local search strategies, which has been reported on other works, but it also shows that our algorithm overcome simple combinations of NSGAII and local search strategies.
We show the performance of MOCGaPBK algorithm optimizing XB _{EB}  XB _{BB} compared with SemiFeaClustMOO [70], MOfuzzy [63], MOGA [34], SOM [71] and Average linkage [72] clustering techniques. All results are exposed for four real life gene expression data sets, i.e., Arabidopsis Thaliana, Yeast Cell Cycle, Yeast Sporulation, and Human Fibroblasts Serum.
To evaluate the compactness and separation of clustering solutions, in Table 5, we show mean values of silhouette index over 20 runs of different algorithms for the four datasets. Here, values in bold represent maximum silhouette index values, revealing that our method achieves better results than existing techniques in all datasets.
To detect whether MOCGaPBK and the competitive clustering techniques operate similarly or not from statistical point of view, we carry out the Friedman test [73]. In this sense, in Table 6 we perform a ranking to each clustering method regarding the mean silhouette index in each dataset.
The test verifies whether the measured average ranks are significantly different from the mean silhouette rank. The method obtains a pvalue of 0.0033 indicating that the difference in the mean silhouette rank obtained by MOCGaPBK algorithm is significant. In fact, our proposed method has an average rank of 1 since it always obtains the best silhouette values in the experimental datasets, while its closest competitor is SemiFeaClustMOO algorithm with an average rank of 2. Clearly, it indicates that our approach obtains groups with better values of compactness and separation than competitive clustering techniques.
To visually demonstrate that clusters yield by MOCGaPBK have high coexpression patterns, we use Eisen and cluster profile plot. To do this, first we determine the best solution (regarding silhouette index), comparing all solutions yield by our method. So, we observe that MOCGaPBK has determined that four is the best number of clusters k for all datasets. Such number of clusters k matches with some values of competitive algorithms [34, 74]. In Figs. 6, 7, 8 and 9, we plot clustering solutions with best silhouette index. For instance, for Yeast Sporulation dataset (Fig. 8a), MOCGaPBK identifies as appropriate partitioning k=4. In such figure, it is evident that expression profiles of the genes in each cluster have similar expression profiles generating similar colour patterns throughout the samples. We can also see (Fig. 8b) that expression patterns of the four clusters of genes differ from each other, while the patterns within a cluster are very similar. So, for example, while cluster 1 has high expression level in the first samples and low in the last ones, the cluster 3 behaves in the opposite way. Similarly, cluster 2 presents low expression levels in first samples but high in remaining ones meanwhile the cluster 4 behaves in opposite way. The other three datasets have similar results. Such situations reveal that genes in the groups found by our algorithm are highly coexpressed.
To establish the biological relevance and coherence of a cluster, we performed an annotation enrichment analysis. In Table 4, we reported to each cluster the three most significant GO terms shared by the genes with their pvalue. It reveals that all the clusters of the solutions found by MOCGaPBK have obtained pvalues less than 0.01, i.e., each cluster has associated biological processes and thus, they are biologically significant and functionally enriched. In this aspect, MOCGaPBK outperforms competitive algorithms since they fail in finding biologically related clusters in some cases. For instance, in Yeast Sporulation dataset, MOGA, FCM, SOM and Average linkage present at least one cluster without biological significance [63].
Conclusions
In this paper we have presented a multiobjective gene clustering algorithm called MOCGaPBK. It includes external biological knowledge during the objective functions optimisation and it integrates intensification and diversification strategies based on both multiobjective PathRelinking and Pareto Local Search.
Results show that MOCGaPBK yields higher quality solutions than other clustering techniques considered here for comparison purposes. It is mainly to the strength of integrating apriori biological knowledge with a multiobjective clustering approach and the use of intensification and diversification strategies. The first one allows having partitions with higher coexpression levels and biological coherence since cluster quality indexes are used to optimise simultaneously gene relationships at expression level and biological functionality. The other aims to improve the clustering solutions to yield higher quality clustering solutions regarding to compactness and separation and to avoid fall into local optima.
The effectiveness of MOCGaPBK was demonstrated quantitatively and visually using statistical comparison test and cluster visualisation tools respectively. Results of silhouette tests, visualisations and annotation enrichment analysis show that the proposed method is able to discover compact, well separated, coexpressed and biologically significant clusters.
To perform the multiobjective clustering process we have used a chromosome representation based on clusters medoid. As a future work we would like to explore a clustering technique based on graph theory to tackle in a better way with datasets where nonconvex groups are present. Furthermore, because we use a multiobjective approach, our algorithm provides a set of solutions, all identically relevant from clustering style point of view. However, for biologists can be more appropriate to have a single solution for clustering of genes or otherwise have a subset of genes that appear grouped together most times. Due to that, in future we would like to develop a voting or ranking technique to identify the set of genes that appear most often in the same clusters, and so facilitate the inference of knowledge by biologists.
Abbreviations
 D _{ BB } :

Biologybased distance
 D _{ EB } :

Expressionbased distance
 Dev:

Overall cluster deviation
 DM:

Decision maker
 GaPBK:

Guided by apriori biological knowledge
 GO:

Gene ontology
 HV:

Hypervolume
 IC:

Information content
 KEGG:

Kyoto encyclopedia of genes and genomes
 MOC:

Multiobjective clustering
 NSGAII:

Nondominated sorting genetic algorithm
 PF:

Pareto front
 PLS:

Pareto local search
 PR:

Pathrelinking
 PS:

Pareto set
 Sep:

Cluster separation
 SGA:

Single objective genetic algorithm
 TR:

Trajectory
 WS:

Wang functional similarity
 XB:

XieBeni index
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Funding
JPA thanks the SENESCYT of Ecuador for its financial support. MIP thanks CEBIB, STICAMSUD 17STIC05 and Proyecto DICYT O61619IP, VRIDEIUSACH. MD thanks CNPq, Alexander von HumboldtStiftung, and the Fundação de Amparo a Pesquisa do Estado do Rio Grande do Sul (FAPERGS)  PRONUPEQ (16/255100005206). This Research was supported by Microsoft under a Microsoft Azure for Research Award.
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The preprocessed data sets, the distance matrices and the best clustering solutions are available in http://bioinformatic.diinf.usach.cl/gapbk/The data and results published here are in part based upon data generated by GOSemSim: http://bioconductor.org/packages/2.6/bioc/html/GOSemSim.htmland the Gene Expression, Genome Variation and Functional Profiling Analysis Suite: http://babelomics.bioinfo.cipf.es/.
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JPA conducted the literature review, developed the code, carried out the experiments, helped interpreting the results and manuscript writing. MD helped interpreting the results and manuscript draft. MIP contributed with the modelling, experiments design, results analysis and manuscript writing. All authors contributed to manuscript refinement and approved the final version.
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ParragaAlava, J., Dorn, M. & InostrozaPonta, M. A multiobjective gene clustering algorithm guided by apriori biological knowledge with intensification and diversification strategies. BioData Mining 11, 16 (2018). https://doi.org/10.1186/s1304001801784
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Keywords
 Multiobjective clustering (MOC)
 Gene expression data
 External biological knowledge
 Pathrelinking (PR)
 Pareto local search (PLS)