 Software
 Open Access
 Published:
lionessR: single sample network inference in R
BMC Cancer volume 19, Article number: 1003 (2019)
Abstract
Background
In biomedical research, network inference algorithms are typically used to infer complex association patterns between biological entities, such as between genes or proteins, using data from a population. This resulting aggregate network, in essence, averages over the networks of those individuals in the population. LIONESS (Linear Interpolation to Obtain Network Estimates for Single Samples) is a method that can be used together with a network inference algorithm to extract networks for individual samples in a population. The method’s key characteristic is that, by modeling networks for individual samples in a data set, it can capture network heterogeneity in a population. LIONESS was originally made available as a function within the PANDA (Passing Attributes between Networks for Data Assimilation) regulatory network reconstruction framework. However, the LIONESS algorithm is generalizable and can be used to model single sample networks based on a wide range of network inference algorithms.
Results
In this software article, we describe lionessR, an R implementation of LIONESS that can be applied to any network inference method in R that outputs a complete, weighted adjacency matrix. As an example, we provide a vignette of an application of lionessR to model single sample networks based on correlated gene expression in a bone cancer dataset. We show how the tool can be used to identify differential patterns of correlation between two groups of patients.
Conclusions
We developed lionessR, an open source R package to model single sample networks. We show how lionessR can be used to inform us on potential precision medicine applications in cancer. The lionessR package is a userfriendly tool to perform such analyses. The package, which includes a vignette describing the application, is freely available at: https://github.com/kuijjerlab/lionessR and at: http://bioconductor.org/packages/lionessR.
Background
Modeling and analyzing biological networks has become an invaluable tool in the analysis of genomic data. While gene expression profiles give us a snapshot of the state of a cell or tissue, network inference algorithms give an estimate of the extent to which genes or gene products interact [1]. Many network inference methods exist [2], most of which require multiple samples and populationlevel data to infer an “aggregate” conditionspecific network [3–8]. These methods first construct a supervised model which then can be applied to single sample data [9–11]; however, they do not directly model networks for individual samples in a population.
We recently developed LIONESS, or Linear Interpolation to Obtain Network Estimates for Single Samples [12], as a way of using populationlevel networks to estimate the corresponding network in each individual sample. LIONESS is based on the idea that each sample has its own network and that each edge in an aggregate network is the “average” (a linear combination) of that edge’s weight across these individual sample networks. LIONESS starts by modeling an aggregate network on an entire population and then removes one sample and rebuilds the network. This is similar to leaveoneout crossvalidation approaches [13]. However, LIONESS then compares the network with and without an individual sample, and uses a linear equation to estimate the network for the withheld sample. Thus, by sequentially leaving out each sample in a population, one can use LIONESS to estimate a network specific to each sample.
The LIONESS equation can be written as:
where \(e_{ij}^{(\alpha)}\) is the weight of an edge between nodes i and j in a network modeled on all (N) samples and \(e_{ij}^{(\alpha q)}\) is the weight of that edge in a network modeled on all samples except the sample of interest (q).
Specifically, LIONESS subtracts edge weights, \(e_{ij}^{(\alpha q)}\), which are derived from a network modeled on all samples except the sample of interest (q), from edge weights, \(e_{ij}^{(\alpha)}\), obtained from the network modeled on all samples; these differences represent the contribution of sample q to the aggregate network. With increasing numbers of samples in the aggregate network model, these contributions become smaller. LIONESS therefore scales these edge weight differences by multiplying them by N, the number of samples that were used to model the aggregate network. Finally, to estimate the single sample edge weights, \(e_{ij}^{(q)}\), LIONESS adds the scaled edge weight differences, \(N\left (e_{ij}^{(\alpha)}  e_{ij}^{(\alpha q)}\right)\), to the edge weights obtained from the network modeled without the sample of interest, \(e_{ij}^{(\alpha q)}\). For more details on how we derived the LIONESS equation, please see the Supplemental Information section published in Kuijjer et al. [12].
LIONESS network estimation is included as an option to use with the PANDA network inference algorithm [7] in our Python tool PyPanda [14]. However, the LIONESS approach is not limited to modeling single sample PANDA networks—it can be used to model single sample networks based on a wide range of network inference algorithms. We developed lionessR, a userfriendly R implementation of LIONESS. The lionessR package can be used to estimate single sample networks for general network methods used in network and cancer biology, including Pearson correlation.
Implementation
We developed the lionessR package in R using CRAN packages devtools and roxygen2. The package depends on R version >=3.0.2 and imports the CRAN library stats. The package is available as opensource code at https://github.com/kuijjerlab/lionessR and can be installed with devtools. Instructions for installation are given on the package’s GitHub site. In addition, an R package is available on Bioconductor at http://bioconductor.org/packages/lionessR.
Within the lionessR package, the lioness() function applies LIONESS (Eq. 1) to the output of a network inference algorithm, as defined by the function netFun(). The default network inference algorithm in netFun() is Pearson correlation, which builds correlation networks by returning an adjacency matrix of Pearson correlation coefficients. We included Pearson correlation as the default function, as correlation has been and continues to be widely used in many network applications [1, 2, 6, 7] and because correlation networks can be modeled on a wide variety of data types. However, netFun() can be substituted with any other uni or bipartite network inference algorithm that returns a complete, weighted adjacency matrix. The lioness() function returns an R data frame that includes weights for all edges in each of the samplespecific networks.
The computation time of lionessR depends on the network reconstruction algorithm used in netFun(). lionessR calculates one aggregate network model based on all N samples, as well as N aggregate network models based on all samples except the sample of interest; therefore its computation time is O(N) times the computation time of modeling a “standard” aggregate network modeled with netFun(). For example, when using the default function netFun() (Pearson correlation) in lionessR on expression data of M genes, it takes O(N·M^{2}) to compute all sample specific networks (if we assume arithmetic operations run in constant time).
The package comes with a vignette that shows how to model networks with lionessR and gives an example of how to analyze single sample lionessR networks. The vignette depends on the CRAN packages igraph and reshape2 and the Bioconductor package limma. The package also includes an example dataset in the object OSdata, which includes expression data for preoperative osteosarcoma biopsies from 53 highgrade osteosarcoma patients, as well as information on whether patients developed metastases within five years since diagnosis of the primary tumor. These data were obtained from the Gene Expression Omnibus (GEO, accession GSE42352), and included samples with at least 70% tumor content and viability, for which RNA was profiled on Illumina human6 v2.0 microarray beadchips and preprocessed using Bioconductor package lumi [15], as previously described [16]. The example data are used in the vignette to model single sample networks for the 53 patients based on correlation networks. The workflow of modeling these individual patient networks and of analyzing them in the context of metastasisfree survival is given in the Results section below.
Results
Application of lionessR to a bone cancer dataset
As an example, we performed an analysis applying lioness() to a gene expression dataset from 53 highgrade osteosarcoma biopsies [16] (Gene Expression Omnibus accession number GSE42352), which is included with the package. Highgrade osteosarcoma is an aggressive primary bone tumor that has peak incidence in adolescents and young adults. About 45% of patients develop metastases, and most metastatic patients eventually die from the disease [17]. We performed a differential correlation network analysis comparing short versus longterm metastasisfree survival (MFS) to understand coregulation differences between the groups and to search for potential therapeutic targets.
For this demonstration, we separated patients into two groups based on those who developed metastases within five years (n=19) and those who did not (n=34). These were the same groups analyzed by Buddingh et al. [18] to compare gene expression levels between short and longterm MFS. To decrease the runtime of our tutorial application, we limited our analysis to the 500 most variable genes based on the standard deviation. We used lioness() to model 53 single sample networks based on Pearson correlation, one for each individual in the population, using the entire population to estimate the background network, with the code:cormat < lioness(dat, netFun),where dat is the input expression data and cormat the lioness output.
Comparative analysis of single sample bone cancer networks modeled with lionessR
We asked whether there were differences in network edge weights between the short and longterm MFS groups. As the aggregate network model in this demonstration is Pearson correlation, a large edge weight in a single sample network indicates that adding that sample increases the Pearson correlation of the aggregate network, while a low edge weight means that addition of the sample decreases the aggregate network’s correlation coefficient for that edge. To reduce the number of statistical tests on these networks (\(500 \choose 2=124750\) potential edges), we modeled two conditionspecific networks and selected those edges that had an edge weight difference of at least 0.5 between these two networks. We then performed a LIMMA analysis [19] to identify those edges whose weights differed significantly between the groups. In parallel, we also used LIMMA to test for significant differences in gene expression levels between groups. We visualized the 50 most significantly perturbed edges (all nominal p<0.001, FDR<0.15) in a network diagram (Fig. 1).
We identified multiple significant differential connections to genes encoding for extracellular matrix proteins, including BGLAP. BGLAP encodes for osteocalcin, a protein secreted by osteoblasts to regulate bone remodeling. BGLAP was connected to both a matrix metalloproteinase (MMP11), involved in breakdown of extracellular matrix, and to genes involved in the immune system—GZMA and HLADQB1. GZMA encodes for Granzyme A, a Tcell and natural killer cellspecific protease, while HLADQB1 is a Matrix Histocompatibility Complex (MHC) Class II gene involved in antigen presentation. All of the edges connected to BGLAP had a moderate to strong negative correlation (range R=[−0.75,−0.61]) in the samples with better MFS, whereas these edges had a weak positive correlation (range R=[0.22,0.26]) in the poor MFS group.
Interestingly, BGLAP was not differentially expressed between these groups (log fold change (logFC) =0.23, p=0.68). This indicates that these processes are tightly regulated in tumors of patients with longterm survival and that loss of this regulation is associated with worse outcome. It also suggests a link between matrix remodeling and recruitment of immune cells, which could indicate that bone remodeling in osteosarcoma may result in the recruitment of immune cells to clear up the cancer, confirming previous findings of osteoclast [20] and macrophage [18] association with MFS in osteosarcoma.
In addition, we identified a highly connected gene, or network “hub,” among the nodes connected to the top 50 edges—STAT1, or Signal Transducer And Activator Of Transcription 1. STAT1 is a transcription factor and thus potentially differentially regulates the target genes with which it is correlated. In fact, all of the edges connected to STAT1 had a moderate to strong negative correlation (range R=[−0.84,−0.42], median R=−0.67) in the samples with better MFS, whereas these edges had a weak to moderate positive correlation (range R=[0.14,0.42], median R=0.30) in the poor MFS group. This suggests that STAT1 may repress expression of these genes in patients with longterm MFS. However, this repression is lost in patients with shortterm MFS. It has been previously shown that in tumors with good prognosis, high STAT1 expression inhibits bone formation [21]. The target genes we identified that connect to STAT1 (Figure 1) were enriched for being annotated to the Gene Ontology term “ossification,” (Fisher’s exact test odds ratio=5.76, pvalue=0.0056), which is consistent with this result. These genes included SOST, SP7, IBSP, IFITM5, and TMEM119.
More importantly, STAT1 is a transcription factor in the interferon signaling pathway—a pathway known to be involved in osteosarcoma, and for which targeted treatment options are available [22]. This indicates that individual patient correlation network analysis with lionessR can pinpoint potential candidates for personalized medicine. Importantly, STAT1 is not differentially expressed itself (logFC =0.44, p=0.19) and neither are many of its target genes. Thus, we would not have been able to obtain this result by analyzing differential expression alone, without placing these genes into a framework of a network. In fact, we previously identified differential gene regulation in the absence of differential expression by analyzing LIONESS networks modeled based on the PANDA [7] network reconstruction framework, which suggested a potential mechanism for sexual dimorphism in colorectal patients [23]. The current example in osteosarcoma highlights the potential of lionessR in modeling networks for individual cancer patients based on other network inference approaches.
Conclusions
Precision medicine uses data about the state of individual genes to match each patient to the therapies that are most likely to be efficacious for them. However, even when therapies target a specific gene mutation, we know that many patients who carry a particular mutation, or whose gene expression signatures correspond to known response biomarkers, do not always respond to targeted treatment. Clearly, to improve precision medicine, we need to better understand the complex relationships that exist between different genes and gene products in individual samples. Networks are a natural way to represent these complex interactions, but methods to infer networks generally “average” over the members of a population. Using networks in precision medicine requires methods that allow inference of network models specific to each individual, reflecting the heterogeneity in the population.
LIONESS represents a method that can fill the gap between methods that infer networks using population data and the need for methods that can model networks specific to each individual. LIONESS estimates individual sample networks by using linear interpolation iteratively, extracting a network for each member of a population [12]. LIONESS essentially measures how removing a single individual from a population changes the aggregate network, and uses those changes to identify the most likely network for that individual. The lionessR package allows users to apply this method in combination with different network inference algorithms, including Pearson correlation.
As an example, we modeled single sample networks based on the 500 genes with the highest variability in expression in an osteosarcoma dataset. We divided this dataset into two groups—patients with either shortterm or longterm MFS. Comparing these two collections of networks using a LIMMA analysis, we identified STAT1 to be significantly coexpressed with a set of “target” genes in biopsies of patients with poor survival. This set of genes was highly associated with biological processes important in osteosarcoma. In addition, STAT1 is part of a biological pathway for which targeted treatment is available. This example highlights how single sample correlation network analysis can be used to inform us on potential precision medicine applications. The lionessR package is a userfriendly tool to perform such analyses.
Availability and requirements
Project name: lionessRProject home page: https://github.com/kuijjerlab/lionessROperating system(s): Platform independentProgramming language: ROther requirements: The vignette walkthrough requires the following R packages: devtools, igraph, reshape2, limmaLicense: CCBY4.0Any restrictions to use by nonacademics: None
Availability of data and materials
All data analyzed in this study are available in the lionessR package on GitHub: https://github.com/kuijjerlab/lionessR
Abbreviations
 LIONESS:

Linear Interpolation to Obtain Network Estimates for Single Samples
 MFS:

Metastasisfree survival
 PANDA:

Passing Attributes between Networks for Data Assimilation
References
 1
Sonawane AR, Platig J, Fagny M, Chen CY, Paulson JN, LopesRamos CM, DeMeo DL, Quackenbush J, Glass K, Kuijjer ML. Understanding tissuespecific gene regulation. Cell Rep. 2017; 21(4):1077–88.
 2
Marbach D, Costello JC, Küffner R, Vega NM, Prill RJ, Camacho DM, Allison KR, Aderhold A, Bonneau R, Chen Y, et al. Wisdom of crowds for robust gene network inference. Nature Methods. 2012; 9(8):796.
 3
Margolin AA, Nemenman I, Basso K, Wiggins C, Stolovitzky G, Dalla Favera R, Califano A. ARACNE: an algorithm for the reconstruction of gene regulatory networks in a mammalian cellular context. BMC Bioinformatics. 2006. Mar 20;7 Article number S7.
 4
Faith JJ, Hayete B, Thaden JT, Mogno I, Wierzbowski J, Cottarel G, Kasif S, Collins JJ, Gardner TS. Largescale mapping and validation of Escherichia coli transcriptional regulation from a compendium of expression profiles. PLoS Biol. 2007; 5(1):8.
 5
Chiquet J, Smith A, Grasseau G, Matias C, Ambroise C. Simone: Statistical inference for modular networks. Bioinformatics. 2008; 25(3):417–8.
 6
Langfelder P, Horvath S. WGCNA: an R package for weighted correlation network analysis. BMC bioinformatics. 2008; 9(1):559.
 7
Glass K, Huttenhower C, Quackenbush J, Yuan GC. Passing messages between biological networks to refine predicted interactions. PloS one. 2013; 8(5):64832.
 8
VillaVialaneix N, Vignes M, Viguerie N, Cristobal MS. Inferring networks from multiple samples with consensus lasso. Quality Technol Quanti Manag. 2014; 11(1):39–60.
 9
HuynhThu VA, Irrthum A, Wehenkel L, Geurts P. Inferring regulatory networks from expression data using treebased methods. PloS one. 2010; 5(9):12776.
 10
Haury AC, Mordelet F, VeraLicona P, Vert JP. TIGRESS: trustful inference of gene regulation using stability selection. BMC Syst Biol. 2012; 6(1):145.
 11
Nicolle R, Radvanyi F, Elati M. CoRegNet: reconstruction and integrated analysis of coregulatory networks. Bioinformatics. 2015; 31(18):3066–8.
 12
Kuijjer ML, Tung M, Yuan G, Quackenbush J, Glass K. Estimating samplespecific regulatory networks. iScience. 2019; 14:226–40.
 13
Allouche D, CiercoAyrolles C, De Givry S, Guillermin G, Mangin B, Schiex T, Vandel J, Vignes M. A panel of learning methods for the reconstruction of gene regulatory networks in a systems genetics context. In: Gene Network Inference. SpringerLink Editors: Alberto de la Fuente: 2013. p. 9–31.
 14
van IJzendoorn DG, Glass K, Quackenbush J, Kuijjer ML. PyPanda: a Python package for gene regulatory network reconstruction. Bioinformatics. 2016; 32(21):3363–5.
 15
Du P, Kibbe WA, Lin SM. lumi: a pipeline for processing Illumina microarray. Bioinformatics. 2008; 24(13):1547–8.
 16
Kuijjer ML, Peterse EF, van den Akker BE, Briairede Bruijn IH, Serra M, MezaZepeda LA, Myklebost O, Hassan AB, Hogendoorn PC, CletonJansen AM. IR/IGF1R signaling as potential target for treatment of highgrade osteosarcoma. BMC Cancer. 2013; 13(1):245.
 17
Buddingh EP, Anninga JK, Versteegh MI, Taminiau AH, Egeler RM, van Rijswijk CS, Hogendoorn PC, Lankester AC, Gelderblom H. Prognostic factors in pulmonary metastasized highgrade osteosarcoma. Pedia Blood Cancer. 2010; 54(2):216–21.
 18
Buddingh EP, Kuijjer ML, Duim RA, Bürger H, Agelopoulos K, Myklebost O, Serra M, Mertens F, Hogendoorn PC, Lankester AC, et al. Tumorinfiltrating macrophages are associated with metastasis suppression in highgrade osteosarcoma: a rationale for treatment with macrophage activating agents. Clin Cancer Res. 2011; 17(8):2110–9.
 19
Ritchie ME, Phipson B, Wu D, Hu Y, Law CW, Shi W, Smyth GK. limma powers differential expression analyses for RNAsequencing and microarray studies. Nucleic Acids Res. 2015; 43(7):47.
 20
EndoMunoz L, Cumming A, Rickwood D, Wilson D, Cueva C, Ng C, Strutton G, Cassady AI, Evdokiou A, Sommerville S, et al. Loss of osteoclasts contributes to development of osteosarcoma pulmonary metastases. Cancer Res. 2010; 70(18):7063–72.
 21
Tajima K, Takaishi H, Takito J, Tohmonda T, Yoda M, Ota N, Kosaki N, Matsumoto M, Ikegami H, Nakamura T, et al. Inhibition of STAT1 accelerates bone fracture healing. J Ortho Res. 2010; 28(7):937–41.
 22
Buddingh EP, Ruslan SEN, Berghuis D, Gelderblom H, Anninga JK, Hogendoorn PC, Egeler RM, Schilham MW, Lankester AC. Intact interferon signaling in peripheral blood leukocytes of highgrade osteosarcoma patients. Cancer Immun, Immunother. 2012; 61(6):941–7.
 23
LopesRamos CM, Kuijjer ML, Ogino S, Fuchs CS, DeMeo DL, Glass K, Quackenbush J. Gene regulatory network analysis identifies sexlinked differences in colon cancer drug metabolism. Cancer Res. 2018; 78(19):5538–47.
Acknowledgements
The authors would like to thank John Platig for helpful discussions.
Funding
This work was supported by grants from the Norwegian Research Council, Helse SørØst, and University of Oslo through the Centre for Molecular Medicine Norway (NCMM) and from the US National Heart, Lung, and Blood Institute of the National Institutes of Health (R01HL111759, P01HL105339, K25HL133599, 1R35CA220523). No specific funding was received for this specific study. The funding bodies had no impact on planning, collection, analysis or interpretation of the data, nor on writing of the manuscript.
Author information
Affiliations
Contributions
Conceptualization, Formal Analysis, Visualization, Writing—Original Draft: MLK; Data Curation, Software: MLK, PHH; Methodology, Investigation: MLK, KG; Supervision: MLK, JQ, KG; Resources, Writing—Review & Editing: all authors, Funding Acquisition: MLK, JQ, KG. All authors read and approved the final manuscript.
Corresponding author
Correspondence to Marieke L Kuijjer.
Ethics declarations
Ethics approval and consent to participate
Not applicable.
Consent for publication
Not applicable.
Competing interests
The authors declare that they have no competing interests.
Additional information
Publisher’s Note
Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Rights and permissions
Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.
About this article
Cite this article
Kuijjer, M.L., Hsieh, P., Quackenbush, J. et al. lionessR: single sample network inference in R. BMC Cancer 19, 1003 (2019). https://doi.org/10.1186/s1288501962357
Received:
Accepted:
Published:
Keywords
 Algorithms
 Software tools
 Computational biology
 Biological networks
 Network analysis
 Coexpression
 Gene regulation
 Precision medicine
 Osteosarcoma