
Research article

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Incorporating timedelays in SSystem model for reverse engineering genetic networks
BMC Bioinformaticsvolume 14, Article number: 196 (2013)
Abstract
Background
In any gene regulatory network (GRN), the complex interactions occurring amongst transcription factors and target genes can be either instantaneous or timedelayed. However, many existing modeling approaches currently applied for inferring GRNs are unable to represent both these interactions simultaneously. As a result, all these approaches cannot detect important interactions of the other type. SSystem model, a differential equation based approach which has been increasingly applied for modeling GRNs, also suffers from this limitation. In fact, all SSystem based existing modeling approaches have been designed to capture only instantaneous interactions, and are unable to infer timedelayed interactions.
Results
In this paper, we propose a novel TimeDelayed SSystem (TDSS) model which uses a set of delay differential equations to represent the system dynamics. The ability to incorporate timedelay parameters in the proposed SSystem model enables simultaneous modeling of both instantaneous and timedelayed interactions. Furthermore, the delay parameters are not limited to just positive integer values (corresponding to time stamps in the data), but can also take fractional values. Moreover, we also propose a new criterion for model evaluation exploiting the sparse and scalefree nature of GRNs to effectively narrow down the search space, which not only reduces the computation time significantly but also improves model accuracy. The evaluation criterion systematically adapts the maxmin indegrees and also systematically balances the effect of network accuracy and complexity during optimization.
Conclusion
The four wellknown performance measures applied to the experimental studies on synthetic networks with various timedelayed regulations clearly demonstrate that the proposed method can capture both instantaneous and delayed interactions correctly with high precision. The experiments carried out on two wellknown reallife networks, namely IRMA and SOS DNA repair network in Escherichia coli show a significant improvement compared with other stateoftheart approaches for GRN modeling.
Introduction
The availability of genome wide expression data has significantly increased interest in systems biology, in particular, reverseengineering gene regulatory networks (GRNs). While static expression data allows the learning of only the network structure, i.e., transcription factors (TF) and target genes interactions, timecourse data allows the modeling of detailed system dynamics over time. In our view, amongst different ways for classification [15], methods for reverseengineering GRNs can be broadly categorized into six major groups, namely (i) coexpression network, (ii) Bayesian network, (iii) differential equation based approach, (iv) regression based approach, (v) meta approaches combining two or more different methods, and (vi) approaches that are based on other principles. Coexpression networks [6, 7] are coarsescale, simplistic models that employ pairwise association measures, such as the partial correlation or conditional mutual information, for inferring the interactions between genes. These methods have low computational complexity and thus can easily scale up to very large networks of thousands of genes [8], but lack a mechanism for modeling system dynamics. Bayesian networks (BN) are more sophisticated models based on the strong foundation of probability and statistics, in which the dependencies between nodes are represented using directed edges and conditional probability distributions. A temporal form of BN, i.e., dynamic Bayesian network (DBN), allows the modeling of system dynamics in discrete time.
In this paper, we focus on differential equation (DE) based approaches, which belong to a sophisticated and well established class of methods for modeling biochemical phenomena, including GRNs [913]. A salient feature of all DEbased approaches is their ability to accurately model system dynamics in continuous time. Of the several linear and nonlinear types of DE models employed for reconstructing GRNs, the SSystem model has gained popularity recently [1419]. Originating from the pioneering work of Savageau [20], the SSystem has been considered as an excellent balance between model complexity and mathematical tractability: it is complex enough to represent a wide range of dynamics, yet is simple enough to allow certain analytical studies.
In GRNs, almost all genetic interactions are invariably delayed. Furthermore, these delayed interactions may have different time lags [21]. Time delays in regulatory interactions are due to the time required for the regulator gene to express its protein product and for the transcription of the target genes to be affected by these regulatory proteins. More specifically, this is the time required for the translation, folding, nuclear translocation, turnover for the regulatory protein, and elongation of the target gene mRNA. For example, in mammals, the transcriptional regulatory timedelays can be from several minutes to several tens of minutes, and are composed of two components: the TF translation/posttranslational processing/translocation time (∼10.5±4 mins), and the target gene transcription and posttranscription processing time (∼2040 mins) [22]. The instantaneous and timedelayed interactions among genes in a toy 3gene network are illustrated in Figure 1. As we can see, gene G_{3} instantaneously regulates genes G_{1} and G_{2}, while G_{1} has a 1unittime delayed regulation on G_{2}.
Most existing approaches for modeling GRNs attempt to capture instantaneous (nontemporal) interactions only. This is the case for all coexpression based approaches and static Bayesian networks, which do not differentiate between static and timecourse expression data. There have been previous attempts for modeling timedelayed genetic interactions with dynamic Bayesian network using timecourse data, such as the method proposed by Zou and Conzen [21], and also our recently proposed method GlobalMIT [23] and [24] (which we call BITGRN2 throughout this paper, as [24] is the improved version of BITGRN). The Recursive Neural Network (RNN) based methods [2529], capable of interpreting complex temporal behavior of gene expression data, have the ability to work with timedelays. However, this timedelay issue is either not welladdressed [28, 29] or the delays are fixed for most of the existing approaches [2527]. Further, so far RNN based methods are incapable of presenting regulations in the degradation phase, which is an inherent feature of SSystem model. The ordinary differential equation (ODE) based methods [911] are limited to work with instantaneous interactions and are incapable of inferring timedelayed regulations. On the other hand, a delay differential equations (DDE) based model was employed in [12], that works with delay, but the time delay parameters were set manually rather than via learning from data. Kim et al.[13] proposed a DDE based method that is capable of working with timedelays. However, the method is limited to working with fixed delays, which are either set to their apriori known values, or otherwise initialized randomly then fixed during the optimization. To the best of our knowledge, there is no differential equation based approach available that can model timedelayed and instantaneous interactions simultaneously, with the flexibility to adapt the delay parameters through optimization.
The main contributions of this paper are twofold. First, it proposes a novel timedelayed SSystem model based on a set of delay differential equations (DDE) which is capable of simultaneously capturing both  timedelayed and instantaneous interactions. Further, it incorporates time delay parameters which are not restricted to take only integer values (corresponding to time stamps in the data) as possible in other discretetime approaches (e.g., dynamic BN), but they can take fractional values. This allows the model to capture the time delays in genetic interactions with higher accuracy, because in reality, the amount of delay takes continuous value. Second, to overcome the limitations of previous optimization approaches, our new search algorithm is designed systematically exploiting the sparse and scalefree nature of GRNs to effectively narrow down the search space. Compared to the existing two SSystem based modeling approaches [16, 19], the proposed approach learns the parameters more accurately despite an increase in the number of model parameters to be learnt. Experimental studies on two synthetic and two real genetic networks show a significant improvement over recently proposed modeling techniques.
Background
Traditional SSystem model
For a network of N genes, the existing SSystem model is given by the following set of ordinary differential equations (ODEs):
Here, for any i^{th} gene, X_{ i } is the expression level, {α_{ i },β_{ i }}’s are the rate constants, and { g_{ i j },h_{ i j }}’s are the kinetic orders. The term ${\alpha}_{i}\prod \underset{j}{\overset{{g}_{\mathit{\text{ij}}}}{X}}$ models the process of RNA synthesis/production, while the term ${\beta}_{i}\prod \underset{j}{\overset{{h}_{\mathit{\text{ij}}}}{X}}$ models the process of RNA degradation. To infer a GRN of N genes using the SSystem model, 2N (N + 1) parameters must be estimated. To reduce computational complexity, method of [30] approximated the original problem as N decoupled subproblems, each having 2(N + 1) parameters. In the i^{th} subproblem corresponding to the i^{th} gene, the parameter set Ω_{ i } = {α_{ i },β_{ i },{g_{ i j },h_{ i j }}_{j = 1 …N}} is estimated by solving the following decoupled SSystem equation:
For solving Eqn. (2), only Y_{ i } = X_{ i } is computed by numerical integration, while ${Y}_{j}={\hat{X}}_{j},\forall j\ne i$, where ${\hat{X}}_{j}$ is obtained by a direct estimation based on the observed expression data of the j^{th} gene [15]. For direct estimation, the commonly used technique of linear spline interpolation [31] can be applied. Although this approximation may decrease the accuracy slightly, the significantly reduced computational burden allows the optimization process to converge to better solutions in much shorter time.
Model evaluation criteria
In order to assess the goodness of SSystem models, previous works commonly employed the squared relative error (SRE) as criterion for model evaluation. As the parameters for each gene in the decoupled systems are learned independently of the others, the SRE for i^{th} gene is given as:
Here t denotes a specific timestamp (TS) in the observed time series of T sample points. ${X}_{i}^{\mathit{\text{cal}}}\left(t\right)$ and ${X}_{i}^{\mathit{\text{exp}}}\left(t\right)$ denote the calculated and observed expression value of gene i at timestamp t respectively. It is to be noted that if the data set consists of several separate time series, then the SRE criterion can simply be extended by summing over all the available time series. Due to decoupling, this SRE criterion for each gene can be minimized independently. The solution for this optimization problem is normally dense, i.e., it has many nonzero parameter values corresponding to many regulators for each gene. However, it is widely reported that GRNs are sparse in nature, and in fact follow a scalefree topology [32, 33]. Thus, a regularization term, similar to LASSO regression, is often added. Authors of [15] were the first to propose a penalty term for model complexity (Eqn. (1) of the supplementary document (Additional file 1)), which was subsequently improved by Noman and Iba [17, 34] as follows:
with K_{ i j } being the kinetic orders of genei sorted in ascending absolute values, I being the maximum number of regulators allowed for each gene, and c being the penalty constant. In this paper, we are referring to Eqn. (4) as regularized squared relative error (RSRE) as it is essentially a regularized version of Eqn. (3). The limitations of the RSRE criterion are: i) although promoting sparse solutions, it still encourages every gene to take on several regulators, since up to I regulators can be taken, free of any penalty, ii) I is a global parameter applied to all genes. Since the number of potential regulators for different genes are different, it is preferable to have the maximum indegree parameter being set adaptively and specifically for each gene, and iii) the penalty weight c is fixed, and thus there is no mechanism for dynamically prioritizing its two objectives (i.e., the two RHS terms) during the optimization. This prioritization is important because, for example, during initial stages of optimization, it is necessary to have emphasis on reducing error, i.e., improving model accuracy (first term), while in the later stages, the emphasis shifts towards reducing model complexity (second term). Our recently proposed evolutionary search [19], unlike previous methods with fixed I, was able to continuously adjust both the value of I (max indegree) and J (min indegree) for every gene based on population statistics:
Here, Z_{ Count } is the total number of nonregulations for the i^{th} gene ( = 2N  total regulations) and, C_{ i } is the scaling factor for the i^{th} gene defined as:
Here, r_{ i } is the number of transcription factors (total regulations) for genei. Details about this fitness function are available in [19] and a brief discussion is included in Section 1.2 of the supplementary document (Additional file 1). Although, the penalty graph generated by the model complexity part resembles the property of powerlaw formalism, the addition of another fractional term in the prefixes of the exponential term (J/r_{ i } and r_{ i }/I) makes the penalty term asymmetric. While a preliminary study [19] on this fitness criteria showed improvement, the applied penalty function being adhoc is not well justified.
Methods
The proposed timedelayed SSystem model
Modeling timedelayed interactions
The traditional SSystem described in Eqn. (1) is a set of ordinary differential equations (ODE), in which the rate of change of a gene expression at a specific time instant t is affected by its own and all other genes’ expression values at that instant. In other words, the model is not versatile to capture time delayed interactions which invariably occur in all biological systems. To do this, it is necessary to replace the system dynamics represented by ODE in Eqn. (1) with delay differential equations (DDE). Let us consider a DDE of the following form:
with the delay parameter τ ∈ [ 0,∞). However, as the rate of change of system response is affected by its previous values at time (t  τ), in practice, τ∈ [ 0,t_{ max }), where t_{ max } is the timespan of the microarray time series experiment. The TimeDelayed SSystem (TDSS) model with a single and fixed delay (τ) can be represented as follows:
In SSystem models with N genes, there can be 2 × N × N regulations, where any of them can be a timedelayed regulation. Hence, we generalize Eqn. (8) in the following form: ?
Here, ${X}_{j,t{\tau}_{\mathit{\text{ij}}}}$ denotes the value of gene X_{ j } at time t  τ_{ ij }, with the delay parameter τ_{ ij }∈ [ 0,τ_{ max }], where τ_{ max } is the maximum possible delay in the considered network. Note that there are two time delay parameters: ${\tau}_{\mathit{\text{ij}}}^{g}$ corresponding to the production part and ${\tau}_{\mathit{\text{ij}}}^{h}$ corresponding to the degradation part of SSystem. The delay matrices are represented as follows:
For both these matrices, {$0\le \{{\tau}_{i,j}^{g},{\tau}_{i,j}^{h}\}\le {\tau}_{\mathit{\text{max}}}$}, ∀_{i,j = 1 …N} and τ_{ max } is the maximum allowed delay of the network.
At any time, the production and degradation rate of the i^{th} gene is affected by its own and other genes’ concentration level at their corresponding delays. If a delay τ_{ ij }, corresponding to an interaction (g_{ ij }/ h_{ ij }), is 0, we have an instantaneous interaction (provided that there is a regulation between genes i and j), whereas a nonzero value of τ_{ ij } gives a delayed interaction. Thus, the proposed TimeDelayed SSystem (TDSS) model is capable of capturing both time delayed and instantaneous genetic interaction in GRNs.
Model parameters
For the traditional SSystem model, in the i^{th} subproblem corresponding to the i^{th} gene, the 2N + 2parameter set Ω_{ i } = {α_{ i },β_{ i },{g_{ ij },h_{ ij }}_{j = 1…N}} needs to be estimated. In the TimeDelayed SSystem model, apart from these parameters, we also have to estimate the 2N timedelay parameters ${\{{\tau}_{\mathit{\text{ij}}}^{g},{\tau}_{\mathit{\text{ij}}}^{h}\}}_{j=1\dots N}$. Thus, a 4N + 2parameter set ${\Omega}_{i}=\{{\alpha}_{i},{\beta}_{i},{\{{g}_{\mathit{\text{ij}}},{h}_{\mathit{\text{ij}}},{\tau}_{\mathit{\text{ij}}}^{g},{\tau}_{\mathit{\text{ij}}}^{h}\}}_{j=1\dots N}\}$ needs to be learned. For learning the timedelay parameters, we follow a twostage approach. First, we employ the Pearson correlation coefficient (PCC) technique to identify the most probable lag of the interaction between any pair of genes. For doing this, we use linear spline interpolation to intrapolate additional data points between any two actual measurements. For a given data set, the maximum time delay (τ_{ max }) permissible for the system is set by considering common regulation time scale (ranging within tens of minutes [22]) and the data sampling rate. Although the proposed TDSS is capable of dealing with any resolution of fractional delay, in this paper we have limited the minimum timedelay step size to 1/10 of the time between two timestamps, provided that the data are regularly sampled. Else, the timedelay step size is set to 1/10 of the time between two closest timestamp in nonregularly sampled data. While using PCC, we fix the expression profile of a regulator gene and shift the target gene’s expression profile forward incrementally one step at a time (minimum timedelay step). The time lag maximizing PCC is considered as the most probable time lag between these two genes. These most probable lag values are then used to initialize the time delay parameters for the evolutionary optimization phase.
Time responses
In the traditional SSystem model, numerical integration is normally performed with the wellknown fourth order RungeKutta method (RK4). For the TimeDelayed SSystem model, we adapt the traditional RK4 method for DDE which takes into account the time delay parameters as described in detail in [35]. For the adapted RK4, initial samples of the regulator gene’s expression profile of length τ_{ max } will be designated as history information, which reduces the available sample size for training. It should be noted that the step size for RK4 integration is set at a small value, allowing the numerical integration to capture the system dynamics accurately. Again, we use linear spline interpolation to generate a continuous history profile. A detailed description of the modified RK4 is presented in Sec. 2.3 in the supplementary document (Additional file 1).
Inference mechanism
Due to the intractable nature of optimization problem, SSystem parameter learning is commonly carried out via evolutionary computation (EC), namely Genetic Algorithm (GA) or Differential Evolution (DE). Recently, DE and its variants, such as trigonometric differential evolution (TDE), have been used extensively because of their versatility [18, 19, 3638]. As an optimization tool for learning model parameters, both DE and TDE perform better than the other conventional evolutionary computation approaches [18, 19]. In this paper, we employ a new TDE approach for learning TDSS parameters. We also employ the Multistage Refinement Algorithm (MRA) [19] as a pruning mechanism for eliminating the weak regulations from the resulting network. Details related to TDE, initial population generation, and MRA are presented in Sec. 1.3 and Sec. 2.4 of the supplementary document (Additional file 1).
Model evaluation criterion
To address various limitations of the regularized squared relative error of Eqn. (4) presented in Sec. 2, we propose a novel fitness function referred to as adaptive squared relative error (ASRE) and given below:
Here, r_{ i } is the total number of actual regulators. B_{ i } is a balancing factor which is used to maintain desired balance between the two terms of ASRE. C_{ i } is the penalty factor for the i^{th} gene, defined as:
with I and J being the maximum and minimum indegree respectively. Note that in our formulation, r_{ i } and I are restricted to be smaller than or equal to N, since a transcription factor generally does not affect both its target gene’s production and degradation simultaneously. In our ASRE criterion, in contrast to a fixed weighting factor c as in Eqn. (4), the penalty factor C_{ i } takes the form of an inverse powerlaw function. This is motivated by the fact that biological networks often have a scalefree structure, in which the node connectivity degree x distributes according to a powerlaw distribution, P(x) ∝ x^{γ}, with the scaling parameter γ ∈ [ 2,3] for various networks in nature, society and technology [33]. Gene regulatory networks generally have low indegrees, with the number of genes having high indegree diminishing according to a powerlaw form. Note that in our formulation, we also enforce a minimum indegree J, thus genes with the number of indegree falling inbetween the minmax number of indegree [J,I] are not penalized (C_{ i } = 1), while genes falling out of this region are penalized according to an inverse power law term (C_{ i } = 1 + d^{γ}, where γ = 2 and d is the number of missing or violated regulations). Sec. 2.4.2 and 2.4.3 in the supplementary document (Additional file 1) explain how our algorithm adaptively adjusts the [J,I] region during the optimization process.
Salient features
We highlight the salient features of the proposed optimization framework as follows:

(i)
Adaptive regulator set size: Our algorithm adaptively and continually adjusts the values of the minmax indegree region [J,I]. Initially, we set J = 0 and I = a value less than or equal to N based on the size of the network. Then, for every l generations, we examine the smallest and largest indegree within the population respectively and set these as new values for J and I.

(ii)
Adaptive balancing factor B_{ i }: The balancing factor B_{ i } is included in Eqn. (12) to dynamically balance the terms corresponding to the network accuracy and the model complexity. For the first initial tens of generations, we set the value of B_{ i } to zero, i.e., we emphasize on network quality first. This allows the algorithm to quickly improve the network accuracy as there are no constraints on complexity. We allow the algorithm to proceed in this manner either until a fixed n_{ e } generations are executed or until the squared relative error is smaller than a specified threshold γ_{ i }. When the individuals in the population achieve stability and improved accuracy, the value of B_{ i } is updated as follows: from the top 50% individuals in the population, we calculate the average network accuracy ANA (first term of Eqn. (12)) and the average model complexity AMC (second term of Eqn. (12), i.e., 2N/(2Nr_{ i })), then set B_{ i } = ANA/AMC. With this, effect of the network accuracy is maintained in ‘balance’ with model complexity. Next, we replace the worst 50% individuals with randomly initialized individuals, and the optimization continues with the value of B_{ i } computed as above.
While our preliminary studies reported earlier [19] also used adaptation of I and J, the implementation was rather adhoc, and had static weight factor. The proposed model evaluation criteria represented by Eqn. (12) and Eqn. (13) are thus novel and perform systematic adaptation of I and J while also simultaneously carrying out adaptive balancing of network complexity and accuracy.
Results and discussions
The proposed TDSS model is evaluated experimentally using both synthetic and reallife networks. As the model parameters increase quadratically with the network size, large scale modeling with the SSystem based models remains a longstanding challenge. For this reason, like previous research on the SSystem [16, 19, 3941], we mainly test our method on small and medium sized networks. We employ two synthetic network studies of different sizes, i.e., a small network with 5 genes and a 20gene medium sized network. For reallife network studies, we present experiments on two small networks, namely IRMA that contains 5 genes, and SOS DNA Repair Network in Escherichia coli containing 8 genes.
With synthetic networks, we investigate network configurations having no delays (instantaneous interactions only) and also in the presence of delays (both instantaneous and timedelayed). For each of these configurations, along with noise free data, we have considered three different levels of Gaussian noise. The four wellknown performance measures [24, 42] namely sensitivity (S_{ n }), specificity (S_{ p }), precision (P_{ r }) and Fscore (F) have been applied for network evaluation. For the methods with executable code available, namely ALG [16, 34] and REGARD [19], we run the respective programs on our generated data. For other methods where no code is available, we extract the performance measure values from their respective original publications for comparison where possible.
With reallife networks, for IRMA, the comparison is carried out with 7 other approaches, namely, ALG [16, 34], REGARD [19], BITGRN2 [24, 42], BANJO [43], TDARACNE [44], NIR and TSNI [45], ARACNE [7]. For the E. coli network, the performance has been evaluated with ALG [34], REGARD [19], STree based approach [39], two approaches from Kimura et al.[40, 46], and several BN based approaches, e.g., [47], BANJO [43], BITGRN2 [24, 42] and GlobalMIT [23]. In addition, the timeresponses of the inferred networks are compared with the actual time expression profiles to show the accuracy of the proposed model in capturing gene expression dynamics. All the inferred timeresponses are shown for the best inference result (in terms of the objective function value, out of 5 separate runs) with error bars indicating the 95% confidence interval (CI).
The proposed algorithm is implemented in C++ using a 2.16 GHz Dualcore CPU PC with 3 GB of RAM. This code is made available upon request. The parameter values for the TDE algorithm were set as follows: Mutation Factor F_{ o } = 0.5, Trigonometric Mutation Factor F_{ t } =0.05, Crossover Factor CF = 0.8, population size Pop = 100. The number of generations when B_{ i } =0 is set to n_{ e } =50 and the specified threshold γ_{ i } to half the value of ASRE of the best individuals in initial population. Once B_{ i } is reset, the indegrees are updated with ARGC algorithm (details in Sec. 2.4.3 of Additional file 1) in every l = 50 generations. The pruning factor ψ = 0.25 (details in Sec. 2.4.5 of Additional file 1) is used in both the stages of Multistage Refinement Algorithm (MRA). For synthetic network, M = 10 datasets are used for reverseengineering, generated for each network from 10 different initial conditions. We have executed the proposed optimization method with TDSS for 1000 generations in the first phase while in the the second phase, MRA is executed for 250 generations. The maximum time delay value (τ_{ max }) was set to 3 timestamps (TS) for all the synthetic networks, as the maximum delay among all delayed regulations was manually set to 3TS for synthetic data generation. For the IRMA networks, τ_{ max } was set to 100 minutes, equivalent to 10TS. For the E. coli network, we set τ_{ max } to 1h, which is also 10TS. The experiments are carried out with 5 separate runs of TDSS with different random initializations for each network. In the following, the best case result represents the best solution of these 5 separate runs, in terms of the objective function value, i.e., the adaptive squared relative error (ASRE) in Eqn. (12).
Synthetic networks
Small scale synthetic network
We investigate the widely studied 5gene synthetic network, first proposed in [14], with the relevant network parameters given in Table 1. From this base network topology, we have generated three different configurations for testing: one network with no delay (Conf1) and two with delays (Conf2 and Conf3). The networks for all three configurations are shown in Figure 2(ac), while the time delay parameter values are shown in Table 2. In all three cases, we have evaluated the performance of TDSS with and without the presence of noise in data.
A. Network with nodelay (Conf1)
In this configuration, all the delay parameters are set to zero. In addition, the methods were also evaluated on noisefree data, as well as data generated with three different levels of Gaussian noise (5%, 10%, and 25%). The performance metrics (S_{ n }, S_{ p }, P_{ r }, F) for the nondelayed network in noisefree setting and with three different levels of noise are shown in Table 3. The proposed method successfully inferred all the regulations (S_{ n } = 1) even in the presence of 25% noise level. Moreover, the other three metrics for TDSS are also very close to the optimal value. It should be noted that, compared to other methods reported in Table 3 the four performance metrics for TDSS are on par with several methods and better than most others. Figure 3(ad) show the timeresponses of a particular gene for all four levels of noise. In particular, we have selected gene1, which exhibits significant changes in expression value over the time course. In addition to detecting all the regulations correctly, the inferred timeresponses are very close to the target expressions. The timeresponses for another gene (gene2) along with the inferred parameters for TDSS (best case result for noisefree data) are shown in Sec. 3 in the supplementary document (Additional file 1). The inferred network for noisefree data (best case) is shown in Figure 2(d).
B. Networks with delay (Conf2 and Conf3)
The delay parameters (i.e., τ^{g} and τ^{h}) for the two timedelayed network configurations (Conf2 and Conf3) are shown in Table 2. For Conf2, 5 out of 13 arcs were randomly chosen and applied a delay of 1TS. For Conf3, we randomly selected six arcs and assigned random fractional delays (in step of 0.1TS) within [0, 3] TS. The delays used in the latter configuration (Conf3) are designed to validate the method on networks having fractional delays. Similar to the nodelay configuration, we have tested the performance of TDSS with noisefree data and also with three different levels of noise. The four performance metrics for TDSS along with three other existing methods are shown in Table 4. While the previous SSystem based methods ALG [34] and REGARD [19], and the BN based approach BANJO [43] considered all inferred edges as instantaneous, TDSS was able to not only infer and segregate these interactions correctly as instantaneous or timedelayed, but the delays were found to be in close agreement to the actual values. The slight deviations between the predicted delays and their actual values might be due to effect of decoupling SSystem equations, and also due to the approximation of data with linear spline interpolation. Since the minimum delay possible is 0.1TS, it is reasonable to accept a deviation of ±0.1TS from its predicted value. From this point of view, an instantaneous interaction in original network appearing with a delay of 0.1TS should be deemed accurate. We observe that, in the presence of noise, all the performance measures of TDSS clearly outperform ALG [34], REGARD [19], and BANJO [43]. The timeresponses for gene1 in all conditions are shown in Figure 4(ad) and Figures 5(ad) for Conf2 and Conf3, respectively. From the four performance metrics and timeresponses for TDSS, it is apparent that the proposed method is very efficient in detecting both instantaneous and delayed regulations, as well as accurately capturing gene expression dynamics. In Sec. 3 of the supplementary document (Additional file 1), the time responses for one more gene and the best case parameter sets inferred by TDSS on noisefree data for both Conf2 and Conf3 are shown. The inferred networks for Conf2 and Conf3 from noise free data (best case) are shown in Figure 2(e) and 2(f), respectively.
Medium scale synthetic network
We now study a medium scale 20gene synthetic network investigated in [34] and [24]. This network has 20 selfinhibitions in the degradation phase and 26 regulations in the production phase. The target kinetic order and rate constant parameters are shown in Table 5. We investigate two different configurations of this network: one with instantaneous regulations only (Conf4), and another with both instantaneous and timedelayed interactions (Conf5). The two different configurations are shown in Figure 6, with their respective delay parameters shown in Table 6.
A. Network with nodelay (Conf4)
From Table 7, it can be observed that, for noisefree and 5%noise in data, the proposed technique successfully inferred all the regulations. While TDSS missed a few regulations with higher levels of noise, all the performance measures are observed to be the best among all considered methods. We show the actual and inferred expression dynamics for gene15, which exhibits high variation throughout the time course, in Figure 7(ad) for all the four noise levels. The time responses for another selected gene (gene18) are shown in Sec. 3 of the supplementary document (Additional file 1). The inferred parameter set for the selective genes on this configuration (for noise free data) are also listed in Sec. 3 in the supplementary document (Additional file 1).
B. Network with delay (Conf5)
This configuration is generated in a similar manner to Conf3 of the 5gene synthetic network, with 8 randomly assigned delayed interactions. The experimental results for this configuration are shown in Table 7. The three existing methods ALG [19, 34], and BANJO [43] do not handle timedelayed regulations, hence considered all the inferred regulations as instantaneous. Due to the presence of timedelayed regulations, all existing methods missed various true regulations (both instantaneous and timedelayed). On the other hand, for noisefree data, TDSS has successfully recovered the true regulations of the target network. In presence of noise, TDSS performance gradually degraded, but still significantly outperformed the three other techniques. Figure 8(ad) show the target and inferred expression dynamics for gene15. The time responses for another gene (gene8) are shown in Sec. 3 of the supplementary document (Additional file 1). The inferred parameter set for the selective genes on Conf5 in noise free condition are also listed in Sec. 3 in the supplementary document (Additional file 1).
Reallife biological networks
The IRMA network
We now consider the wellstudied IRMA network, a reallife invivo synthetic network constructed within the Saccharomyces cerevisiae yeast [12]. This is a small scale network composed of five genes (CBF1, GAL4, SWI5, GAL80, ASH1) having a total of 8 regulations. Two gene expression data sets were collected from [12]: the ON data set corresponds to the shifting of the growth medium from glucose to galactose, while the OFF data set corresponds to shifting from galactose to glucose. In the ON (OFF) dataset, there are 16(21) timesamples which were evenly sampled every 20(10) minutes respectively. For the sake of uniformity with the OFF dataset, we have intrapolated (using linear spline interpolation) an additional data point between every two samples in the ON dataset to make it a uniform 10minute sampled data set. Moreover, as in [39], we also consider the presence of selfinhibition in degradation phase for each genes (i.e., h_{i,i} > 0). It is noted that the mutual interactions between GAL4 and GAL80 are proteinprotein interactions, which, in principle, are not reflected in gene expression data. Thus, Cantone et al. [12] also considered a ‘simplified’ network by combining GAL4 and GAL80, and ignoring their mutual regulations. The IRMA original and simplified networks are shown in Figure 9(a) and Figure 9(d), respectively.
The experimental results for the IRMA network are shown in Tables 8 and 9. We note that, for the ON data set, TDSS was successful in inferring a higher number of true regulations (11 out of 13) than any other methods reported in this paper. As a result, the sensitivity is highest amongst all the methods (S_{ n } = 0.85), while the other performance metrics S_{ p } and P_{ r } are very close to the best values. More importantly, the Fscore (F), which is the harmonic mean of precision and sensitivity, is relatively high for TDSS (second best). While considering the simplified network, although the performance metrics of TDSS are not the best among the methods, they are still very competitive and close to the best values. The inferred network with only true regulations for both original and simplified structure are shown in Figure 9(b) and Figure 9(c), respectively. On the OFF data set, TDSS also exhibits good performance, with the best S_{ n } and F among all considered methods. Further, on the simplified network, all the four performance measures are found best for the TDSS. The TDSS time responses for all genes on the ON data sets in Figure 10 clearly indicate that the inferred gene expressions are very close to the corresponding targets.
Additionally, we highlight here an interesting biological finding made during the computational analysis. In particular, the proposed SSystem model was successful in uncovering the important timedelay interaction in the IRMA network for the activation of CBF1 from SWI5. More specifically, from observations during the invivo experiment, this regulation was experimentally characterized as a timedelayed interaction of 100 minutes [12]. The proposed SSystem model is the firstever method that discovered, not only this timedelayed nature of the interaction, but also the accurate timedelay value (in minutes). In particular, for the IRMAON data set, the SWI5 →CBF1 interaction was inferred as a 94minute delayed regulation. The same regulation was also successfully inferred as a delayed regulation of 92 minutes while reconstruction was performed with the IRMAOFF dataset. Both the delay values are very close to the original time delay value of 100 minutes as reported in [12]. All the inferred true regulations along with corresponding timelags are listed in Table 10. Indeed, we believe that this interesting finding is made possible due to the novel features present in the proposed TDSS model.
The SOS DNA repair network in Escherichia coli
Next, we consider the wellstudied SOS DNA repair network within Escherichia coli (E. coli). While the entire DNA repair system of E.coli involves more than 100 genes [39, 47], only its 30 genes contribute towards key regulations at the transcription level. We make use of the expression data set collected by Ronen et al.[52], which contains information about 8 genes namely uvrD, lexA, umuD, recA, uvrA, uvrY, ruvA, and polB. The data sets are obtained from four different experiments under various UV light conditions, with the gene expression levels being measured at 50 instants evenly spaced at a 6minute interval. Following [34, 40, 46], we normalize the input data by dividing the expression profile of each gene by its maximum value. Historically, there were two versions of this SOS network in the literature, one involving 6 genes (uvrD, lexA, umuD, recA, uvrA and polB) [19, 39, 46], and another involving all the 8 genes [23, 24, 43, 47], both inferred from Ronen et al.’s expression data [52]. Herein, we study both the networks.
As the exact ground truth for this network is not precisely known, it is not possible to calculate the four performance metrics, i.e., sensitivity, specificity, precision and Fscore. However, from the functional description of each gene in the original paper [52], it is generally recognized that suppressions of all genes from lexA and activation to lexA from recA are considered as true regulations. On the 6gene SOS network, TDSS successfully inferred 6 out of these 7 known regulations, missing only the activation recA →lexA. Authors in [19, 39, 46] also considered this 6gene SOS network and successfully inferred 5, 6, and 5 true regulations, respectively, as detailed in Table 9. The method of Kimura et al. [53] inferred all the 7 known regulations, however, with all incorrect regulatory signs. For the 8gene SOS network, ALG [34] and several BN based approaches, namely BANJO [43], Perrin et al. [47], GlobalMIT [23], and BITGRN2 [24] respectively inferred 6, 2, 4, 5, 4 true regulations. The proposed TDSS successfully inferred 7 known regulations, as detailed in Table 11. For the 8gene SOS network, all the methods considered in this comparison, including TDSS, failed to infer the regulation lexA →ruvA.
It should be noted that, other than the known regulations reported in Table 11, considered as true positives, the proposed TDSS also inferred some unknown regulations. These can be either novel regulatory interactions, or false positive findings. These interactions are shown as “Novel Interactions” in Table 11. We refer to the existing stateoftheart methods where these unknown regulations were justified. For example, the regulation of lexA by umuD was previously discovered and discussed in [47] and [16],[34]. This regulation was also discovered by two of our previously proposed methods REGARD [19] and GlobalMIT [23]. This regulation is inferred by the proposed TDSS on both the 6gene and 8gene networks. Further, the regulation uvrA ⊣lexA was also inferred by TDSS for both networks. This interaction was also previously reported in [47] and [53]. Finally, the regulation uvrA ⊣recA was inferred by 4 existing methods [23],[39],[43],[47], while TDSS did not discover this connection. Historically, all these three novel regulations mentioned in Table 11 were first reported by Perrin et al.[47], and later rediscovered by other methods [16],[43]. However, for confirming the biological validity of these interactions, suitable wetlab experiments are yet to be performed. It is noted that for TDSS and other SSystem based methods, selfregulations in either or both the production or degradation phase is normally needed to balance the model. However we clarify that, selfregulations in DE based approaches reflect the selfdependency of a gene expression upon its own value at a previous time point, rather than a physical selfinteraction.
For the 6gene (8gene) SOS network, the proposed TDSS method was successful in inferring 8 (9) “true”+“novel” regulations, including 4 (5) regulations which were reported as timedelayed. These results indicate the presence of possible delayed regulations in the network. All the inferred true regulations are shown in Table 12 with their corresponding timelags. The true regulations inferred correctly in TDSS for both the subnetworks are shown in Figure 11(a) and Figure 11(b). Further, Figure 12 shows that, despite the inherent noise in reallife data, TDSS time responses for all the 6 genes are very close to the target expression patterns.
Computational efficiency
Finally, we consider the issue of computational time. We have compared the timing of TDSS with two other SSystem based approaches, namely REGARD [18] and ALG [34]. The average times for these three methods to infer the parameters of a single gene on seven networks considered in this paper are shown in Figure 13. We observe that, despite a significant increase in the number of parameters to model the time delay, TDSS was found to converge much faster than ALG [34], and marginally faster than REGARD [18]. This demonstrates the benefit of dynamically adapting the regulatory genes cardinality (i.e., minimum J and maximum I indegrees) as explained in the proposed Methods section. The adaptation of I and J narrows down the search space significantly and speeds up convergence. In Figure 14, we show the optimization process for gene1 of Conf1 (5gene network) in a particular run.
Conclusion
Timedelayed regulations are an inherent characteristics of all biological networks. While there have been some recent efforts using Bayesian network (BN) approach to simultaneously model timedelayed and instantaneous interactions, the current state of the art SSystem approaches cannot model timedelayed interactions. In this paper, we have proposed a novel method to incorporate timedelayed interactions in the existing SSystem modeling approaches for reverse engineering genetic networks. The proposed Timedelayed SSystem (TDSS) model is capable of simultaneously representing both instantaneous and timedelayed regulations. Apart from the kinetic order and rate constant parameters as in traditional SSystem models, additional parameters for the time delays are necessary for TDSS full description. To make the optimization effective and efficient in the increased parameter space, we proposed a novel objective function based on the sparse and scalefree nature of genetic network. The inference method was also redesigned, based on adaptive systematic adaptation of the max and min indegrees for gene cardinality, and systematic balancing between time response accuracy and network complexity during the optimization process. The RK4 numerical integration technique has also been suitably adapted for TDSS. Investigations carried on small and medium synthetic networks with various levels of noise, as well as on two reallife genetic networks show that our approach correctly captures the timedelayed interactions and outperforms other existing SSystem based methods.
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Acknowledgements
This work is supported in part by NICTA (National Information and Communication Technology Australia) research in Systems Biology flagship program.
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Authors’ contributions
ARC, MC and NXV developed the concepts and drafted the manuscript. ARC developed the algorithms and carried out the experiments. MC and NXV suggested the biological data, experiments and provided biological insights on the results. MC provided overall supervision, direction and leadership to the research. All authors read and approved the final manuscript.
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Keywords
 Bayesian Network
 Delay Differential Equation
 Synthetic Network
 Supplementary Document
 True Regulation