BayesCCE: a Bayesian framework for estimating cell-type composition from DNA methylation without the need for methylation reference

Sep 1, 2018

https://genomebiology.biomedcentral.com/articles/10.1186/s13059-018-1513-2

Abstract
We introduce a Bayesian semi-supervised method for estimating cell counts from DNA methylation by leveraging an easily obtainable prior knowledge on the cell-type composition distribution of the studied tissue. We show mathematically and empirically that alternative methods which attempt to infer cell counts without methylation reference only capture linear combinations of cell counts rather than provide one component per cell type. Our approach allows the construction of components such that each component corresponds to a single cell type, and provides a new opportunity to investigate cell compositions in genomic studies of tissues for which it was not possible before.

Background
DNA methylation status has become a prominent epigenetic marker in genomic studies, and genome-wide DNA methylation data have become ubiquitous in the last few years. Numerous recent studies provide evidence for the role of DNA methylation in cellular processes and in disease (e.g., in multiple sclerosis [1], schizophrenia [2], and type 2 diabetes [3]). Thus, DNA methylation status holds great potential for better understanding the role of epigenetics, potentially leading to better clinical tools for diagnosing and treating patients.

In a typical DNA methylation study, we obtain a large matrix in which each entry corresponds to a methylation level (a number between 0 and 1) at a specific genomic position for a specific individual. This level is the fraction of the probed DNA molecules that were found to have an additional methyl group at the specific position for the specific individual. Essentially, these methylation levels represent, for each individual and for each site, the probability of a given DNA molecule to be methylated. While simple in principle, methylation data are typically complicated owing to various biological and non-biological sources of variation. Particularly, methylation patterns are known to differ between different tissues and between different cell types. As a result, when methylation levels are collected from a complex tissue (e.g., blood), the observed methylation levels collected from an individual reflect a mixture of its methylation signals coming from different cell types, weighted according to mixing proportions that depend on the individual’s cell-type composition. Thus, it is challenging to interpret methylation signals coming from heterogeneous sources.

One notable challenge in working with heterogeneous methylation levels has been highlighted in the context of epigenome-wide association studies (EWAS), where data are typically collected from heterogeneous samples. In such studies, we typically search for rows of the methylation matrix (each corresponding to one genomic position) that are significantly correlated with a phenotype of interest across the samples in the data. In this case, unless accounted for, correlation of the phenotype of interest with the cell-type composition of the samples may lead to numerous spurious associations and potentially mask true signal [4]. In addition to its importance for a correct statistical analysis, knowledge of the cell-type composition may provide novel biological insights by studying cell compositions across populations.

In principle, one can use high-resolution cell counting for obtaining knowledge about the cell composition of the samples in a study. However, unfortunately, such cell counting for a large cohort may be costly and often logistically impractical (e.g., in some tissues, such as blood, reliable cell counting can be obtained from fresh samples only). Due to the pressing need to overcome this limitation, development of computational methods for estimating cell-type composition from methylation data has become a key interest in epigenetic studies. Several such methods have been suggested in the past few years [5–10], some of which aim at explicitly estimating cell-type composition, while others aim at a more specific goal of correcting methylation data for the potential cell-type composition confounder in association studies. These methods take either a supervised approach, in which reference data of methylation patterns from sorted cells (methylomes) are obtained and used for predicting cell compositions [5], or an unsupervised approach (reference-free) [6–10].

The main advantage of the reference-based method is that it provides direct (absolute) estimates of the cell counts, whereas, as we demonstrate here, current reference-free methods are only capable of inferring components that capture linear combinations of the cell counts. Yet, the reference-based method can only be applied when relevant reference data exist. Currently, reference data only exist for the blood [11], breast [12], and brain [13], for a small number of individuals (e.g., six samples in the blood reference [11]). Moreover, the individuals in most available data sets do not match the reference individuals in their methylation-altering factors, such as age [14], gender [15, 16], and genetics [17]. This problem was recently highlighted in a study in which the authors showed that available blood reference collected from adults failed to estimate cell proportions of newborns [18]. Furthermore, in a recent work, we showed evidence from multiple data sets that a reference-free approach can provide substantially better correction for cell composition when compared with the reference-based method [19]. It is therefore often the case that unsupervised methods are either the only option or a better option for the analysis of EWAS.

As opposed to the reference-based approach, although can be applied for any tissue in principle, the referencefree methods do not provide direct estimates of the cell-type proportions. Previously proposed reference-free methods allow us to infer a set of components, or general axes, which were shown to compose linear combinations of the cell-type composition [8, 9]. Another more recent reference-free method was designed to infer cell-type proportions; however, as we show here, it only provides components that compose linear combinations of the cell-type composition rather than direct estimates [10]. Unlike cell proportions, while linearly correlated components are useful in linear analyses such as linear regression, they cannot be used in any nonlinear downstream analysis or for studying individual cell types (e.g., studying alterations in cell composition across conditions or populations). Cell proportions may provide novel biological insights and contribute to our understanding of disease biology, and we therefore need targeted methods that are practical and low in cost for estimating cell counts.

In an attempt to address the limitations of previous reference-free methods and to provide cell count estimates rather than linear combinations of the cell counts, we propose an alternative Bayesian strategy that utilizes prior knowledge about the cell-type composition of the studied tissue. We present a semi-supervised method, BayesCCE (Bayesian Cell Count Estimation), which encodes experimentally obtained cell count information as a prior on the distribution of the cell-type composition in the data. As we demonstrate here, the required prior is substantially easier to obtain compared with standard reference data from sorted cells. We can estimate this prior from general cell counts collected in previous studies, without the need for corresponding methylation data or any other genomic data.

We evaluate our method using four large methylation data sets and simulated data and show that our method produces a set of components that can be used as cell count estimates. We observe that each component of BayesCCE can be regarded as corresponding to scaled values of a single cell type (i.e., high absolute correlation with one cell type, but not necessarily good estimates in absolute terms). We find that BayesCCE provides a substantial improvement in correlation with the cell counts over existing reference-free methods (in some cases a 50% improvement). We also consider the case where both methylation and cell count information are available for a small subset of the individuals in the sample, or for a group of individuals from external data. Notably, existing reference-based and reference-free methods for cell-type estimation completely ignore this potential information. In contrast, our method is flexible and allows to incorporate such information. Specifically, we show that our proposed Bayesian model can leverage such additional information for imputing missing cell counts in absolute terms. Testing this scenario on both real and simulated data, we find that measuring cell counts for a small group of samples (a couple of dozens) can lead to a further significant increase in the correlation of BayesCCE’s components with the cell counts.

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