iASeq: integrative analysis of allelespecificity of proteinDNA interactions in multiple ChIPseq datasets
 Yingying Wei†^{1},
 Xia Li†^{2, 3},
 Qianfei Wang^{2} and
 Hongkai Ji^{1}Email author
DOI: 10.1186/1471216413681
© Wei et al.; licensee BioMed Central Ltd. 2012
Received: 15 June 2012
Accepted: 20 November 2012
Published: 29 November 2012
Abstract
Background
ChIPseq provides new opportunities to study allelespecific proteinDNA binding (ASB). However, detecting allelic imbalance from a single ChIPseq dataset often has low statistical power since only sequence reads mapped to heterozygote SNPs are informative for discriminating two alleles.
Results
We develop a new method iASeq to address this issue by jointly analyzing multiple ChIPseq datasets. iASeq uses a Bayesian hierarchical mixture model to learn correlation patterns of allelespecificity among multiple proteins. Using the discovered correlation patterns, the model allows one to borrow information across datasets to improve detection of allelic imbalance. Application of iASeq to 77 ChIPseq samples from 40 ENCODE datasets and 1 genomic DNA sample in GM12878 cells reveals that allelespecificity of multiple proteins are highly correlated, and demonstrates the ability of iASeq to improve allelic inference compared to analyzing each individual dataset separately.
Conclusions
iASeq illustrates the value of integrating multiple datasets in the allelespecificity inference and offers a new tool to better analyze ASB.
Keywords
Allelespecific binding Transcription factor Histone modification Data integration Nextgeneration sequencing Statistical modelBackground
In a diploid organism, each somatic cell has two copies of the genome. At certain genomic loci, gene expression, DNA methylation, transcription factor (TF) binding or histone modification (HM) can be allelespecific. In other words, the two alleles can behave differently. These phenomena, also known as allelespecific expression (ASE), allelespecific DNA methylation (ASM) and allelespecific binding (ASB, including both allelespecific TF binding and allelespecific histone modifications), can contribute to phenotypic diversity and may play important roles in adaptive evolution [1–3]. Many allelespecific (AS) events have been found to correlate with variants in genomic sequences [4–11]. Comprehensively characterizing allelespecificity therefore can help with linking genotypes to phenotypes. Abnormal AS events have also been linked to various diseases [12–15]. For instance, loss of imprinting in IGF2 has been associated with increased risk of colorectal cancer [12]. This again highlights the importance of studying allelespecificity.
Early methods for analyzing AS events rely on lowthroughput technologies such as real time quantitative PCR [1]. Later, application of SNP arrays has made the AS analysis highthroughput [16–19]. More recently, the rapidly evolving highthroughput sequencing technologies opened the door to produce digital readout of AS events genomewide without being constrained by any specific array design [5, 14, 15, 20–24]. This brings many new opportunities as well as analytical challenges.
ChIPseq, a technology that couples chromatin immunoprecipitation with highthroughput sequencing, has become the stateoftheart approach for mapping genomewide TF binding sites and HMs [25–28]. However, so far the value of this technology for studying ASB has not been fully utilized. Detecting ASB from a single ChIPseq dataset often suffers from low statistical power. This is because only a small fraction of reads in each ChIPseq sample are mapped to heterozygote SNPs, and only these reads are informative for inferring allelespecificity. To make the ChIPseq based ASB analysis more useful, it is important to have either experimental or analytical innovations to increase the power for detecting allelespecificity.
ChIPseq data in public domains grow rapidly. A recently developed database hmChIP, for instance, has compiled over 450 human and mouse ChIPseq datasets representing approximately 2000 samples from 140+ different TFs and HMs [29, 30]. The large volume of data provides a new opportunity to improve detection of ASB. Conceptually, an integrative analysis of ChIPseq data for different TFs and HMs from the same individual and cell type may allow one to discover the synergistic correlation patterns of allelespecificity among different proteins. These correlation patterns can then be utilized to integrate information from multiple datasets to improve the ASB detection. For example, if the allelic imbalance of TF A and HM B always cooccur, then analyzing their ChIPseq data jointly will increase the effective number of reads available for allelic inference which will then increase the statistical power. Unfortunately, existing data analysis tools cannot deal with this emerging opportunity. Methods available for analyzing ASE or ASB using the nextgeneration sequencing data are all designed for analyzing one dataset at a time. While a few methods are developed for solving problems such as read mapping biases [31], construction of individualized genome sequences [32], and combining multiple SNPs in the same gene to infer ASE [33], no methods and software tools are available for jointly analyzing multiple ChIPseq datasets together to discover synergy patterns of allelespecificity among multiple proteins and then use the correlation patterns to increase the power of ASB detection by borrowing information across datasets.
In this article, we present an integrated solution to this problem by developing a new approach, iASeq, for jointly analyzing allelespecificity in multiple ChIPseq datasets. iASeq uses a Bayesian hierarchical mixture model to describe unknown correlation patterns of allelespecificity among multiple datasets. These patterns can be discovered automatically from the data by fitting the model using an ExpectationMaximization (EM) algorithm. Using the identified correlation patterns, the model allows one to integrate information from multiple datasets to improve the ASB detection. Applying this approach, we analyzed 40 ENCODE [34] ChIPseq datasets in GM12878 cells, representing a total of 77 samples from 34 TFs and HMs. The analysis demonstrates the ability of iASeq to automatically integrate information from multiple datasets to significantly improve the detection of allelic imbalance. iASeq is implemented as an R package which is freely available from Bioconductor [35].
Methods
Data structure
After read mapping and data preprocessing (see Additional file 1: Supplemental Methods S.1), we count reads for each allele at each heterozygote SNP. For SNP i, dataset d and replicate sample j, let x_{ idj } and y_{ idj }be the read counts for the reference allele and nonreference allele respectively. Let n_{ idj }=x_{ idj } + y_{ idj } be the total read count (See Figure 1a for a toy example). ProteinDNA binding can be skewed to the reference allele (SR), skewed to the nonreference allele (SN), or not allelespecific (NS). We use a binary variable b_{ id } to indicate whether SNP i is SR (b_{ id }=1) or not (b_{ id }=0) in dataset d. If b_{ id }=1, then SNP i is assumed to be SR in all replicate samples in dataset d. Similarly, we introduce another binary indicator c_{ id } to indicate whether SNP i is SN or not in dataset d. b_{ id }and c_{ id } cannot be equal to one at the same time. If b_{ id }=0 and c_{ id }=0, then SNP i is NS in dataset d. The configuration at each SNP i can be described by two vectors B_{ i }=(b_{i 1},⋯,b_{ iD }) and C_{ i }=(c_{i 1},⋯,c_{ iD }) (See Figure 1d for a cartoon illustration). Based on these notations, (x_{ idj },y_{ idj }), or equivalently (x_{ idj },n_{ idj }), are the observed data for SNP i in sample (d,j), whereas the indicators b_{ id } and c_{ id } are unobserved.
Main intuition and challenge
Our primary goal is to infer for each SNP whether there is allelic imbalance in each dataset. This is equivalent to inferring b_{ id } and c_{ id }. A simple solution to this problem is to analyze each individual dataset separately, but this approach has low statistical power since the counts (x_{ idj },n_{ idj }) usually are small.
If one knows how different datasets are correlated in terms of allelic imbalance, this knowledge may be used to improve the data analysis. For instance, if the allelic imbalance of two proteins A and B are closely correlated, then observing skewed read counts for protein A will provide information for inferring the allelic imbalance of protein B. Integrating the data from both A and B will increase the effective number of reads available for statistical inference, which will then lead to increased statistical power.
In reality, how different proteins are correlated is usually unknown. However, one may learn it by studying the data from many SNPs. Each SNP has three possible states in each dataset: SR, SN and NS. For D datasets, there are 3^{ D } possible configurations in total. From studying many SNPs, one can know the relative frequencies (or mixing proportions) of these 3^{ D }configurations. The mixing proportions will tell how different datasets are correlated. For instance, let [s_{1},s_{2},⋯,s_{ D }] be the skewness configuration of a SNP in the D datasets. If the mixing proportions for three configurations [NS,NS,⋯,NS], [SR,SR,⋯,SR] and [SN,SN,⋯,SN] are 0.9, 0.05 and 0.05, then no other configurations exist in the data and all datasets are perfectly correlated in terms of the allelic imbalance. In other words, at a particular SNP, if one dataset is SR, then all the other datasets are also SR. If one is SN, then all the others are also SN. On the other hand, if other configurations have nonzero mixing proportions, then not all datasets are perfectly correlated, and at a particular SNP, one allows the possibility that only a subset of datasets are correlated. For instance, if the mixing proportion for a configuration [SR,SR,NS,⋯,NS] is 0.03, then there will be 3% of SNPs that are skewed to the reference allele in the first two datasets but not skewed in the other datasets. Therefore, knowing the mixing proportions of all 3^{ D }configurations will tell one the correlation structure in the data. This knowledge can then be used to improve statistical inference at each individual SNP by facilitating information sharing across datasets. For example, if the configuration [SR, SR, SN] has a much higher mixing proportion than [SR, SR, NS], then observing strong skewness towards the reference allele of a SNP in the first two datasets will imply that, a priori, the SNP is highly likely to be skewed to the nonreference allele in the third dataset and has much lower probability to be nonskewed for both alleles. The principle here is the same as the principle represented by the Bayesian hierarchical models in the statistical literature.
A limitation of this approach is that one has to enumerate all 3^{ D } AS configurations in order to describe the correlation. As the number of datasets increases, the number of possible configurations increases exponentially. Thus this approach does not scale well with the increasing D. Later, in our analysis of GM12878 data, D=40 and 3^{ D }>10^{19}. This simple approach is clearly intractable.
To circumvent the difficulty of documenting the frequencies of all 3^{ D }configurations, iASeq employs a technique that can describe the major correlation patterns in the data using a few probability vectors whose values vary from 0 to 1 rather than being dichotomous (i.e., 0 or 1). This approach significantly reduces the model complexity but keeps the flexibility to account for all 3^{ D }configurations. It is easily scalable to increasing dataset number. The correlation structure in the model can then be used to improve the statistical inference of allelic imbalance at each SNP in each individual dataset.
Probability model
iASeq is based on the Bayesian hierarchical mixture model below that uses several probability vectors to describe the major correlation patterns among multiple datasets (Figure 1). The model assumes that SNPs can be grouped into K + 1 classes with different allelespecificity patterns (K≪3^{ D }), and the observed data are viewed as generated as follows:
● First, a class label a_{ i }is randomly assigned to each SNP i according to a probability vector Π=(Π_{0},Π_{1},⋯,Π_{ K }). Here, Π_{ k }=Pr(a_{ i }=k) is the prior probability to assign a SNP to class k. $\sum _{k}{\Pi}_{k}=1$.
● If the class label a_{ i }=0, then B_{ i }=(0,⋯,0) and C_{ i }=(0,⋯,0). In other words, all SNPs in class 0 are background SNPs, and they are NS in all datasets. If a_{ i }=k and k≠0, then SNP i can be skewed, and its [b_{ id };c_{ id }]s in different datasets are generated independently according to the following probabilities: Pr(b_{ id }=1,c_{ id }=0a_{ i }=k)=v_{ kd }and Pr(b_{ id }=0,c_{ id }=1a_{ i }=k)=w_{ kd }. We assume v_{ kd } + w_{ kd }<1, i.e., Pr(b_{ id }=0,c_{ id }=0a_{ i }=k)=1−v_{ kd }−w_{ kd }>0. The model implies that each class is associated with two vectors of probabilities V_{ k }=(v_{k 1},⋯,v_{ kD }) and W_{ k }=(w_{k 1},⋯,w_{ kD }). For SNPs in class k, B_{ i }and C_{ i }are generated according to the probabilities in V_{ k }and W_{ k }.
● Next, the observed read counts are generated based on the AS configurations specified by B_{ i }s and C_{ i }s. Consider SNP i and dataset d. If b_{ id }=1, then (x_{ idj },n_{ idj }) in each replicate sample (d,j) is generated according to a probability distribution Pr(x_{ idj },n_{ idj }b_{ id }=1,c_{ id }=0)=Pr(n_{ idj }b_{ id }=1,c_{ id }=0)Pr(x_{ idj }n_{ idj },b_{ id }=1,c_{ id }=0)≡Pr(n_{ idj })f_{idj 1}(x_{ idj }). Here we assume that the marginal distribution of n_{ idj }does not depend on b_{ id }and c_{ id }, and we use f_{idj 1}(x_{ idj }) to denote the conditional distribution Pr(x_{ idj }n_{ idj },b_{ id }=1,c_{ id }=0). Data in different replicate samples are assumed to be generated independently. Similarly, if c_{ id }=1, then (x_{ idj },n_{ idj })s are generated according to Pr(x_{ idj },n_{ idj }b_{ id }=0,c_{ id }=1)=Pr(n_{ idj })f_{idj 2}(x_{ idj }). If b_{ id }=0 and c_{ id }=0, then (x_{ idj },n_{ idj })s are generated according to Pr(x_{ idj },n_{ idj }b_{ id }=0,c_{ id }=0)=Pr(n_{ idj })f_{idj 0}(x_{ idj }).
For SNP i and dataset d, we organize data from all replicates j=1,⋯,J_{ d } into X_{ id }=(x_{id 1},⋯,x_{ idJ }_{ d }) and N_{ id }=(n_{id 1},⋯,n_{ idJ }_{ d }). For SNP i, X_{ i }=(X_{i 1},⋯,X_{ iD }) and N_{ i }=(N_{i 1},⋯,N_{ iD }) contain data from all datasets. The final observed data are X=(X_{1},⋯,X_{ I }) and N=(N_{1},⋯,N_{ I }) which are the ensemble of data from all SNPs.
Let A=(a_{1},⋯,a_{ I }) be the collection of class membership indictors of all SNPs, and let B=(B_{1},⋯,B_{ I }) and C=(C_{1},⋯,C_{ I }) be the SR and SN indictors for all SNPs. A, B and C are the unobserved missing data one wants to infer.
Organize the probability vectors V_{ k }and W_{ k } from different classes into two matrices $\left(\right)close="">{\mathit{V}}_{K\times D}={({\mathit{V}}_{1}^{T},\cdots \phantom{\rule{0.3em}{0ex}},{\mathit{V}}_{K}^{T})}^{T}$ and $\left(\right)close="">{\mathit{W}}_{K\times D}={({\mathit{W}}_{1}^{T},\cdots \phantom{\rule{0.3em}{0ex}},{\mathit{W}}_{K}^{T})}^{T}$. V, W, and the probability vector Π that describes the class abundance are the unknown model parameters. K is assumed to be fixed. The choice of K and specification of data generating distributions Pr(n_{ idj }), f_{idj 0}(x_{ idj }), f_{idj 1}(x_{ idj }) and f_{idj 2}(x_{ idj }) will be discussed later.
Based on this model, each SNP class k (k≠0) is associated with two vectors of probabilities V_{ k }and W_{ k } which characterize the allelic imbalance preferences in different datasets for SNPs belonging to class k. For example, if a class has [V_{ k };W_{ k }] = [(0.8,0.7,0.1,0.1); (0.1,0.1,0.8,0.1)], then SNPs in this class have high probability to be SR in datasets 1 and 2, and high probability to be SN in dataset 3, but they have low probability to be allelespecific in dataset 4. Since V_{ k } and W_{ k }are probabilities rather than 01 vectors, each class k can generate all 3^{ D } AS configurations. Therefore, SNPs in the same class are not required to have the same AS configuration (e.g., a class can have one SNP with configuration [SR,SR,NS,NS] while at the same time another SNP with configuration [SR,NS,SR,NS]), although they usually have similar AS configurations because SNPs in the same class are all generated using the same probability vectors. Meanwhile, there are K different classes, and each class has a different [V_{ k };W_{ k }] which specifies a different preference to generate the skewing configurations. Thus, whereas SNPs in the same class tend to have similar [B_{ i };C_{ i }] configurations, SNPs from different classes tend to have very different configurations. Conceptually, this is similar to a modelbased clustering analysis in which SNPs are grouped into K + 1 clusters based on their [B_{ i };C_{ i }] configurations. However, an important difference here is that [B_{ i };C_{ i }]s are unknown.
Our model assumes that [b_{ id };c_{ id }]s of the same SNP in different datasets are a priori independent conditional on the class membership a_{ i }. However, [b_{ id };c_{ id }]s from different datasets are not independent marginally if one integrates out the class label a_{ i }. For example, the marginal probability Pr([b_{ id };c_{ id }]= [1;0])=$\sum _{k}\mathrm{Pr}\left(\right[{b}_{\mathrm{id}};{c}_{\mathrm{id}}]=\phantom{\rule{0.3em}{0ex}}[1;0\left]\right{a}_{i}=k\left)\mathrm{Pr}\right({a}_{i}=k)=$$\sum _{k=1}^{K}{\Pi}_{k}{v}_{\mathrm{kd}}$. On the other hand, the joint probability $\phantom{\rule{0.3em}{0ex}}\mathrm{Pr}\left(\right[\phantom{\rule{0.3em}{0ex}}{\mathit{B}}_{i};{\mathit{C}}_{i}]=[\phantom{\rule{0.3em}{0ex}}(1,1,\phantom{\rule{0.3em}{0ex}}\cdots \phantom{\rule{0.3em}{0ex}},\phantom{\rule{0.3em}{0ex}}1);(0,0,\cdots \phantom{\rule{0.3em}{0ex}},0)\left]\right)\phantom{\rule{0.3em}{0ex}}=\phantom{\rule{0.3em}{0ex}}\sum _{k=1}^{K}{\Pi}_{k}\left(\prod _{d}{v}_{\mathrm{kd}}\right)$, which is clearly different from the product of the marginals $\prod _{d}\mathrm{Pr}\left(\right[{b}_{\mathrm{id}};{c}_{\mathrm{id}}]=[1;0\left]\right)=\prod _{d}\left(\sum _{k=1}^{K}{\Pi}_{k}{v}_{\mathrm{kd}}\right)$. This explains why our model can be used to describe the correlation among multiple datasets despite the conditional independence assumption. Intuitively, if one views the model as a clustering analysis of SNPs based on [B_{ i };C_{ i }], then each cluster will represent a cooccurrence pattern of allelespecificity across multiple proteins. The marginal correlation among multiple datasets is described by multiple clusters, whereas within each cluster the data in different datasets are generated independently. In real data, a small K (i.e., a small number of SNP classes) usually is sufficient to describe the major correlation structure among datasets. Using Π, V and W to describe the correlation among datasets only requires O(KD) parameters, which is significantly less complex than O(3^{ D }) parameters. At the same time, the iASeq model still provides the flexibility to accommodate all 3^{ D } possible [B_{ i };C_{ i }] configurations as all of them have nonzero probability to occur.
Data generating distributions
To fully specify the model, one also needs to specify the data generating distributions Pr(x_{ idj },n_{ idj }b_{ id },c_{ id })=Pr(n_{ idj })Pr(x_{ idj }n_{ idj },b_{ id },c_{ id }). The primary goal of iASeq is to infer whether two alleles are different. We assume that information on allelespecificity is only contained in Pr(x_{ idj }n_{ idj },b_{ id },c_{ id }), and therefore the exact form of Pr(n_{ idj }), i.e., the marginal probability distribution of the total read count, is irrelevant for our purpose. As such, we mainly focus on modeling the conditional distribution of x_{ idj }given n_{ idj }, b_{ id } and c_{ id }, i.e., the three distributions f_{idj 0}(x), f_{idj 1}(x) and f_{idj 2}(x).
iASeq models these distributions hierarchically in two steps. First, x_{ idj } is assumed to follow a binomial distribution x_{ idj }n_{ idj },p_{ idj }∼Bin(n_{ idj },p_{ idj }), where p_{ idj } is the probability that a read generated at SNP i in sample (d,j) represents the reference allele. Next, we model p_{ idj }depending on the values of b_{ id }and c_{ id }.
Here $\left(\right)close="">{C}_{n}^{k}$ is the binomial coefficients “n choose k”, and B(. .) is the beta function.
Joint probabilities and model fitting
Conditional on the observed data, Pr(N) is a constant that does not contain parameters of interest, therefore it is absorbed into a proportionality constant not shown in the formula above. Using this joint posterior, an EM algorithm can be derived to search for posterior mode $(\widehat{\Pi},\widehat{\mathit{V}},\widehat{\mathit{W}})$ of $\mathrm{Pr}(\Pi ,\mathit{V},\mathit{W}\mathit{X},\mathit{N})=\sum _{\mathit{A},\mathit{B},\mathit{C}}\mathrm{Pr}(\mathit{A},\mathit{B},\mathit{C},\Pi ,\mathit{V},\mathit{W}\mathit{X},\mathit{N})$ in which the missing indictors A, B and C are all integrated out (see Additional file 1: Supplemental Method S.4).
For the Dirichlet prior, we use η=2 (see Additional file 1: Supplemental Method S.3 for a discussion on the choice of parameter for the Dirichlet prior). In the EM algorithm, we assume that the class number K is given. In order to choose the optimal K, we run the algorithm multiple times using different values of K. We choose the best K using the Bayesian Information Criterion (BIC) (see Additional file 1: Supplemental Method S.5).
Statistical inference of allelespecificity
Using ${\stackrel{~}{P}}_{\mathrm{id}}$, SNPs can be rank ordered for biologists to choose candidates to design followup studies. For each top ranked SNP, one can determine its skewing direction by comparing Pr(b_{ id }=1,c_{ id }=0X_{ i },N_{ i },Π,V,W) and Pr(b_{ id }=0,c_{ id }=1X_{ i },N_{ i },Π,V,W). The one with the larger value determines the direction. Finally, the posterior probabilities of top N SNPs can be converted to an estimate of false discovery rate (FDR) using $\mathrm{FDR}\left(N\right)=\sum _{i\in \mathrm{top}\phantom{\rule{2.22198pt}{0ex}}N\phantom{\rule{2.22198pt}{0ex}}\mathrm{SNPs}}(1{\stackrel{~}{P}}_{\mathrm{id}})/N$.
Formula 7 shows that two types of information contribute to Pr(b_{ id },c_{ id }X_{ i },N_{ i },Π,V,W): (1) Pr(a_{ i }=kX_{ i },N_{ i },Π,V,W), which is determined using information from all D datasets, and (2) Pr(b_{ id },c_{ id }a_{ i }=k,X_{ i },N_{ i },Π,V,W), which only uses information specific to dataset d conditional on Π, V and W. Thus for each particular dataset d, the datasetspecific information is weighted by information obtained from other datasets to determine the SNP ranking. Intuitively, if allelic imbalance in two datasets are correlated, then observing an AS event in one dataset will suggest that a relatively weak skewing event observed at the same SNP in the other dataset is very likely to be a true AS event. In contrast, if no AS event is observed in one dataset, then a relatively weak skewing event observed at the same SNP in the other dataset is likely to be a false positive. This is the underlying nature of using Pr(a_{ i }=kX_{ i },N_{ i },Π,V,W) to reweigh information in Pr(b_{ id },c_{ id }a_{ i }=k,X_{ i },N_{ i },Π,V,W), and it provides the foundation for improving SNP ranking by borrowing information across datasets. In real applications, Π, V,W are unknown, and they are replaced by the posterior mode obtained from the EM algorithm.
Results
GM12878 data and preprocessing
We collected 40 ENCODE [36] ChIPseq datasets with a total of 77 samples together with a genomic DNA sample in GM12878 lymphoblastoid cells (Additional file 2: Table S1). GM12878 is a female and is one of the most extensively studied cell lines in ENCODE. Within each dataset, the number of replicate samples varied from 1 to 3. We downloaded the raw sequence reads of all 78 samples and mapped them to human genome (hg18) (see details in Additional file 1). We removed repeated sequences from the ChIPseq datasets to avoid PCR duplicates, which may skew the determination of allelic biases. In other words, if multiple reads have exactly the same sequence, only one copy is retained. We obtained the genotype data for GM12878 from [37].
As previously described in [31], there are two different types of read mapping biases that may affect the analysis of AS events: the reference bias and the inherent bias. The reference bias often occurs when one maps sequence reads to a reference genome. If one allows the same number of mismatches in the alignment, a read from the nonreference allele is less likely to be mapped back to the reference genome compared to a read from the reference allele, since the nonreference read has one more mismatch to the reference genome. This phenomenon is known as the reference bias. This type of bias, if it exists, is automatically taken care of by the iASeq model through the parameter p_{dj 0} which models the background skewing probability and is estimated using all reads mapped to heterozygote SNPs in each sample. If there is reference mapping bias, p_{dj 0}will take a value different from 0.5 to adjust for the bias. One may remove reference bias before the analysis by masking SNPs in the reference genome during the alignment or by aligning reads to a diploid personal genome. This situation will also be automatically recognized by iASeq through the estimation of p_{dj 0} from the data (if there is no bias, p_{dj 0}=0. 5). Therefore, regardless of whether the reference bias has been removed from the data in the preprocessing or not, the iASeq model is able to automatically handle it and adjust the inference accordingly.
The intrinsic bias is a different type of bias. As shown by [31], even if the reference bias is removed (e.g., by masking SNPs in the reference genome), the inherent bias still exists. For example, suppose sequence 1 (e.g., xxxAxxx) and sequence 2(e.g., xxxTxxx) are two reads that differ only in one position (i.e., A/T). It is possible that sequence 1 is easier to be mapped back to its correct location in the genome than sequence 2 if the second sequence has many repeats in the genome. This bias reflects the inherent characteristics of the genome and cannot be removed by masking variants in the reference genome or by mapping reads to a diploid personal genome. In the above example, masking A and T in the original reads is also not a solution, since a priori one does not know which position in a read corresponds to a SNP position and therefore should be masked without first aligning the read to the genome. When a heterozygote SNP has inherent bias, one allele will have higher read counts than the other even if the two alleles have the same binding level. To avoid this bias, we used the approach described in [22, 31] to remove SNPs with the inherent bias.
We began with 1,704,166 heterozygote SNPs and filtered out 149,996 (8.8%) SNPs with inherent bias. Next, we eliminated SNPs that were not bound by any TF or associated with any HM in any dataset (see Additional file 1: Supplemental Methods S.1.1, S.1.2 and Additional file 3: Table S2 for details). After applying these filters, 94,519 heterozygote SNPs remained. These 94,519 SNPs were then analyzed by iASeq.
A simulation study
Before we apply iASeq to the real data, we first tested its performance in simulations that took into account real data characteristics. Our simulations kept the same design as the real GM12878 ChIPseq data, with the same number of datasets and the same number of replicates within each dataset, except that the genomic DNA sample was not used here since we knew the truth in the simulations and did not need genomic DNA as a control for potential bias. To create the simulation data, we first applied iASeq to the real GM12878 data to identify 86,353 SNPs that were not skewed in any dataset using Pr(a_{ i }=0X_{ i },N_{ i },Π,V,W)>0. 5 as cutoff. To mimic the real background noise, these SNPs were resampled by a bootstrap procedure to create the background SNPs in the simulations, and we kept the read counts (x_{ idj },n_{ idj }) of each background SNP as is in the simulated data. Next, we simulated ASB SNPs and added them to the background. Simulations were carried out under two different scenarios (Figure 2).
● Scenario 2: Four correlation patterns (classes 14) were created in addition to the background class (class 0). Class 1 and class 2 were the same as in simulation 1. Classes 3 and 4 were two new patterns. SNPs in class 3 were SR in datasets 2140, and SN in datasets 110. Class 4 was the mirror image of class 3. The abundance of the classes 0 to 4 was (0.90,0.025,0.025,0.025,0.025).
Given the simulated [B_{ i };C_{ i }] configurations, we then simulated the read count data for ASB SNPs as described in detail in Additional file 1: Supplemental Methods S.6. Simulations done in this way was able to keep the major characteristics of real data while allowing us to benchmark the performance of different methods since we knew the truth.
 1.
Deviation statistic (d): SNPs were ranked based on x _{ id }/n _{ id }−p _{d 0}. Here we estimated ${p}_{d0}=\frac{1}{{I}^{\prime}}\sum _{i:{n}_{\mathrm{id}}\ne 0}{p}_{\mathrm{id}}=\frac{1}{{I}^{\prime}}\sum _{i:{n}_{\mathrm{id}}\ne 0}\frac{{x}_{\mathrm{id}}}{{n}_{\mathrm{id}}}$, where I ^{ ′ }is the number of SNPs for which n _{ id }≠0.
 2.
Naive z statistic (z): SNPs were ranked based on $\frac{{x}_{\mathrm{id}}/{n}_{\mathrm{id}}{p}_{d0}}{\sqrt{({p}_{d0}\ast (1{p}_{d0})/{n}_{\mathrm{id}})}}$. Here p _{d 0}was estimated as in the deviation statistic d.
 3.
Naive Bayes statistic (b): SNPs were ranked using $\left\right({x}_{\mathrm{id}}+2\ast {\stackrel{~}{p}}_{d0})/({n}_{\mathrm{id}}+2){\stackrel{~}{p}}_{d0}$. Here ${\stackrel{~}{p}}_{d0}=\frac{1}{I}\sum _{i}\frac{{x}_{\mathrm{id}}+2\ast {p}_{d0}}{{n}_{\mathrm{id}}+2}$ where p _{d 0}was estimated as in the deviation statistic d. The implicit assumption here is that x _{ id }p _{ id }∼Bin(n _{ id },p _{ id }) and p _{ id }∼Beta(α _{ d },β _{ d }) with ${\alpha}_{d}=2{\stackrel{~}{p}}_{d0}$ and ${\beta}_{d}=2(1{\stackrel{~}{p}}_{d0})$. The posterior mean of p _{ id }is used to construct the ranking statistic.
 4.
Empirical Bayes statistic (B): SNPs were ranked using $\left\right({x}_{\mathrm{id}}+{\widehat{\alpha}}_{d})/({n}_{\mathrm{id}}+{\widehat{\alpha}}_{d}+{\widehat{\beta}}_{d}){\stackrel{\u030c}{p}}_{d0}$. We estimated ${\stackrel{\u030c}{p}}_{d0}=\frac{{\widehat{\alpha}}_{d}}{{\widehat{\alpha}}_{d}+{\widehat{\beta}}_{d}}$. The implicit assumption is the same as the naive Bayes statistic, but now we estimate α _{ d }and β _{ d }based on the observed data using the method of moments as in iASeq (see Additional file 1: Supplemental Method S.2).
 5.
Single dataset EM (singleEM): We fitted a mixture model of SR, SN and NS with distributions f _{ idjp }(·),p=0,1,2 and mixing probabilities v _{ d }, w _{ d }and 1−v _{ d }−w _{ d }for each dataset d without considering other datasets. SNPs were ranked using a posterior probability similar to ${\stackrel{~}{P}}_{\mathrm{id}}$, but now determined based on information in dataset d only (see Additional file 1: Supplemental Method S.7 for details).
Figure 3 compares the number of true positives, T P_{ d }(q), in the top q SNPs reported by each method in each dataset d. In Figures 3ac and 3eg, T P_{ d }(q) is plotted as a function of q in a few representative datasets. These plots show that iASeq outperformed all singledataset based methods, and it was able to substantially improve the power for detecting allelespecificity.
In general, the observed differences between iASeq and the d, z, b and B statistics could be caused by many factors such as use of different statistical models, ranking statistics, or methods for parameter estimation. However, the comparison between iASeq and the single dataset EM represents a wellcontrolled comparison since these two methods used exactly the same distributional assumptions and parameter estimation methods. The only difference between them was that iASeq used information from multiple datasets whereas singleEM was based on one dataset only. This wellcontrolled comparison shows that jointly modeling multiple datasets is able to improve the allelic inference.
To examine whether iASeq was able to bring improvement in all datasets, we computed the Area under the Receiver Operating Characteristic (ROC) curves (AUC) for each method in each dataset using the top 2000 ranked SNPs. In each dataset, we computed the proportion of improvement in terms of AUC brought by iASeq over the best singledataset based ranking method (i.e., $\mathrm{dAUC}=\frac{\mathrm{AU}{C}_{\mathit{\text{iAseq}}}\mathrm{AU}{C}_{\mathit{\text{bestsingle}}}}{\mathrm{AU}{C}_{\mathit{\text{bestsingle}}}}$). dAUC>0 means iASeq is able to bring improvement. Figures 3d and 3h show the distribution of dAUC across all 40 datasets as a histogram. The results show that iASeq was able to improve the SNP ranking in almost all datasets.
Analysis of real data
Our simulation study demonstrates the ability of iASeq to discover correlation patterns of allelespecificity and improve the detection of skewed SNPs. Next, we applied iASeq to analyze the 41 real datasets (78 samples) in GM12878 cells. In real data, we do not have comprehensive knowledge about the truth. Therefore, unlike simulations, we were not able to assess the FDR estimates. For this reason, we mainly focused on analyzing the correlation patterns of allelespecificity and testing whether iASeq can improve the SNP ranking.
Correlation patterns of allelespecificity
Figures 5b and 5c show the posterior mode of V_{ k }and W_{ k } for the two nonbackground classes. It turned out that these two classes corresponded to two global directions of allelespecificity, SR and SN, respectively. Since the assignment of reference or nonreference allele depends on the reference genome, the assignment per se is not of biological interest. However, recall that GM12878 is a single person, therefore at each single SNP, the nucleotide representing the reference or nonreference allele is the same across all datasets analyzed here. Given this fact, what these results essentially tell is that at each single SNP, most TFs and HMs in our analysis were highly correlated in terms of allelespecificity, and if they are skewed, they tend to be skewed toward the same direction (i.e., the same allele). For instance, for SNPs in class 1, both H3K4me3 (from the Broad Institute) and H3K27ac (Broad) had high probability to be SR, with (v_{ kd },w_{ kd }) equal to (0.9337,0.0070) and (0.9730,0.0041) respectively (Figure 5c). The probability that one is SR and the other one is SN was small. Similarly, for SNPs in class 2, both H3K4me3 and H3K27ac were highly likely to be SN simultaneously ((v_{ kd },w_{ kd })=(0.0061,0.9835) for H3K4me3 (Broad) and (0.0040,0.9897) for H3K27ac (Broad)). While the allelic imbalance of most TFs and HMs were highly correlated, H3K27me3, a HM involved in gene repression, was an exception. In both nonbackground classes, H3K27me3 had much lower skewing probabilities compared to the other proteins (Figure 5c). Within each class, the difference in the skewing probability between the two alleles was also much weaker for H3K27me3 as compared to the other proteins. For instance, in class 1, while most other proteins showed strong preference to be skewed toward the reference allele, H3K27me3 can be skewed to the reference allele at some SNPs and skewed to the nonreference allele at many other SNPs. Therefore, the allelic imbalance in H3K27me3 is not strongly correlated with the allelic imbalance of the other proteins analyzed here. For the genomic DNA which was used as control here, the skewing probabilities (v_{ kd },w_{ kd }) in both classes were fairly low as shown in Figure 5bc. In both classes, the probability for not being skewed in the genomic DNA (i.e., 1−v_{ kd }−w_{ kd }) was bigger than 0.95. This indicates that the high probability of skewing observed in the other datasets was not an artifact.
The coordinated allelic imbalance of different proteins toward the same allele has also been observed in a recent study [38]. In that study, the authors analyzed AS of 24 TFs and found that when multiple TFs bind to the same SNP, they frequently bind to the same allele. Moreover, those authors did not observe any pair of TFs that regularly bind the same position on alternate alleles. Our observation here therefore is consistent with their finding.
Increased power for detecting allelespecificity compared with single dataset analysis
We ranked SNPs based on the posterior probabilities ${\stackrel{~}{P}}_{\mathrm{id}}$ in each dataset. The iASeq ranking was compared with the rankings provided by the five singledataset based methods described above. Since we do not know the truth, we used two types of independent information as gold standard to benchmark the ranking results.
Comparisons with other methods
Most existing studies on allelespecificity were conducted using inhouse data analysis pipelines. A tool developed by Skelly et al. [33] and AlleleSeq [32] are two software tools accessible to thirdparty users for AS analysis. The method proposed by Skelly et al. [33] is designed for analyzing ASE in RNAseq data. It first fits a background model using genomic DNA and then feeds the estimated parameters into a Bayesian model that combines information from multiple SNPs within a gene to infer ASE. When we applied this method to analyzing the GM12878 ChIPseq data, two problems occurred. First, the method uses Markov Chain Monte Carlo (MCMC) to fit the background model from the genomic DNA which, as alerted by [33], is wellknown for its slow speed and difficulties for users to monitor the convergence. Our genomic DNA data had 94,519 SNPs which covered 12,417 genes. Running this algorithm on this data using the parameter settings recommended by [33] on a machine with 2.7 GHz CPU and 4 Gb RAM took more than 60 days. Second, after feeding the background model parameters obtained from the first step to the inference model in the second step, the algorithm stopped execution after a few iterations. This is because the original model was developed for deeply sequenced RNAseq rather than ChIPseq, where the average read count covering a heterozygote SNP in a ChIPseq dataset is only 0.64. As a result, the model developed in [33] did not fit the real data in ChIPseq experiments. This lackoffit caused the program to stop early, likely due to the abnormally fitted parameters causing various computation problems (e.g., overflow). For this reason, although the method proposed by [33] represents an advanced solution for analyzing RNAseq ASE, it cannot be directly used to analyze ASB in ChIPseq data without significantly redesigning the model and algorithm. For this reason, it is not further compared here.
Comparison of iASeq and AlleleSeq
Gold standard  ChrX  All Caltech ASE exonic SNPs  Autosomal Caltech ASE exonic SNPs  

TF  T_{d}  AlleleSeq  iASeq  T_{d}  AlleleSeq  iASeq  T_{d}  AlleleSeq  iASeq 
YaleCFOS  41  3  4  9  5  3  9  5  3 
YaleMYC  122  9  22  39  5  10  38  5  10 
YaleJUND  289  13  31  24  4  8  23  4  7 
YaleMAX  105  3  18  18  3  1  18  3  2 
YalePolIII  25  2  2  0  0  0  0  0  0 
Discussion
Interpretation of the correlation patterns
When analyzing the real data in GM12878, iASeq found two nonbackground AS patterns, representing two opposite directions of allelic imbalance. Since the assignment of reference and nonreference allele depends on the reference genome, whether a SNP is skewed toward reference or nonreference allele per se does not have direct biological meaning. What these two patterns essentially suggest is that the allelic imbalances of multiple proteins at a single SNP are correlated and have high preference to be skewed toward the same allele. In other words, the two patterns should be viewed as a pair and interpreted together.
In general, although one may view different allelic imbalance patterns in iASeq as different clusters of SNPs, these clusters only describe the similarities among SNPs in terms of their skewness directions, rather than the similarities in terms of their functions. The direction is defined using the reference/nonreference allele. The reference or nonreference allele for different SNPs can have different meanings (e.g., for one SNP, the maternal allele may be the reference allele, whereas for another SNP the paternal allele may be the reference allele). Therefore within each cluster, even though SNPs have similar skewness pattern, they are not necessarily functionally related to each other. One should not confuse the SNP clusters here with the clusters obtained from the traditional gene expression microarray data analysis, where coexpressed genes in a cluster often have similar functions. In iASeq, the clusters only serve as a tool to describe the correlation structure among different datasets (i.e., proteins), rather than the functional correlation among different SNPs. The correlation patterns among datasets are used by iASeq to inform one how to integrate information across datasets (i.e., which datasets are highly correlated and therefore can borrow information from each other) to improve detection of AS events for each individual SNP and dataset. In order to understand functions of the detected AS events, one needs to further correlate the iASeq results with other information (e.g., one may determine the parentoforigin of each SNP first and then study various phenomena such as imprinting).
Our observation that different proteins prefer to be skewed in the same direction is consistent with a recent observation reported in [38] that AS of 24 different TFs are skewed toward the same allele. A number of factors could contribute to the observed correlation. First, biologically it is plausible that functionally related HMs and TFs have correlated allelespecificity. For instance, both H3K4me2 and H3K4me3 are markers for active transcription. Therefore, for a specific SNP, if the reference allele is associated with a gene with active transcription but the nonreference allele is not, then it is very likely that both H3K4me2 and H3K4me3 will be skewed toward the reference allele. For another SNP, if the nonreference allele is transcribed but the reference allele is not, then both H3K4me2 and H3K4me3 will have high probability to be skewed toward the nonreference allele. In the genome, H3K4me2 and H3K4me3 are skewed toward reference allele for some SNPs, and skewed toward nonreference allele for some other SNPs. Therefore the skewed SNPs could naturally fall into two clusters, representing two opposite AS directions. Second, as pointed out by [38], the coordinated AS could also occur as a result of the difference in the chromatin landscape between the two alleles. For instance, if the chromatin on one allele is more open and accessible, it could increase the overall binding probability of multiple different proteins, leading to correlated allelic skewing.
While our results show that most analyzed TFs/HMs tend to be skewed toward the same direction, these results do not imply that these proteins are perfectly correlated in terms of allelespecificity at each and every SNP. In iASeq, the correlation patterns V_{ k }and W_{ k }are probabilistic patterns rather than 01 vectors. Each correlation class k can generate all 3^{ D } AS configurations. For instance, for a class with [V_{ k };W_{ k }]= [(0.9,0.9,0.9,0.1); (0.1,0.1,0.1,0.1)], it is possible to have one SNP with configuration [SR,SR,NS,NS] and at the same time another SNP with configuration [SR,NS,SR,NS]. Therefore, SNPs in the same class are not required to have the same AS configuration, even though they tend to have similar AS configurations. The probabilistic patterns are used here to provide a parsimonious description of the complex correlation structure in the data, so that one can circumvent the difficulty of handling 3^{ D } AS configurations whose complexity increases exponentially. As a consequence of using this parsimonious model, multiple weak correlation patterns without strong enough data support could be merged into a bigger class. For instance, consider two AS patterns [V_{ k };W_{ k }]=[(1,1,0,0);(0,0,0,0)] (i.e., [SR,SR,NS,NS]) and [V_{ k };W_{ k }]=[(0,0,1,1);(0,0,0,0)] (i.e., [NS,NS,SR,SR]). Suppose both patterns are equally likely to occur in the data. If each pattern is only associated with a small number of SNPs, then a parsimonious model will prefer merging them together into one single class with [V_{ k };W_{ k }]=[(0. 5,0. 5,0. 5,0. 5);(0,0,0,0)]. For this reason, iASeq only discovers correlation patterns that have sufficient data support so that they can be distinguished from other patterns. It will not report weak patterns, which could be real but do not have enough data support to allow them to be robustly recovered. For users, this means that at the cluster level, they may not be able to see weak but real AS correlation patterns if these patterns are not associated with enough number of SNPs. On the other hand, for the purpose of inferring whether or not each SNP is allelespecific in each dataset, these parsimonious correlation patterns are sufficient for describing the correlation structure in the data and serving as a prior to guide the information sharing across datasets. The information sharing will lead the increased ASB detection power, and the eventual AS configuration at each individual SNP will be determined by the posterior probabilities of (b_{ id },c_{ id }) (i.e., ${\stackrel{~}{P}}_{\mathrm{id}}$) rather than the clusterlevel prior probabilities [V_{ k };W_{ k }]. Therefore, in the final AS calls, the model still allows each SNP to have its own AS configuration which may not necessarily be the same as the AS configurations of other SNPs from the same cluster.
Consistent with [38], in the two nonbackground AS patterns discovered here, proteins skewed toward the same direction did not always correspond to known proteinprotein interactions. As pointed out by [38], this could happen as a result of allelic imbalances of different proteins being caused by a common underlying factor such as allelic difference in chromatin landscape. It could also reflect unknown proteinprotein interactions. For iASeq specifically, there is a third reason, that is, multiple small patterns can be merged into a bigger probabilistic class as described before. For example, because of the use of probabilistic patterns, two patterns [SR,SR,NS,NS] and [NS,NS,SR,SR] may be merged into a single SNP class (e.g., V_{ k };W_{ k }=[(0. 5,0. 5,0. 5,0. 5);(0,0,0,0)]). As a result, only looking at the pattern represented by V_{ k };W_{ k }, one cannot tell the details of proteinprotein interactions, such as these interactions only exist between datasets 1 and 2, or between 3 and 4, but not between the other pairs of datasets. What one can tell from this merged pattern is that, when the allelic imbalance occurs in these four datasets, they will be skewed toward the same direction, i.e., the reference allele in this example.
In summary, while the correlation patterns in iASeq provide some insights on the correlation of allelic imbalance among different datasets, one should not overinterpret them. The primary goal of these patterns is to describe the correlation structure in the data so that information from different datasets can be shared in a principled way to increase the power of statistical inference. This also points to an important difference between this study and previous studies that reported coordinated allelespecificity among multiple proteins. The previous studies only reported the correlation as a biological finding, but did not provide a statistical method to further utilize the correlation structure to improve the statistical inference. In contrast, iASeq provides a general and rigorous statistical method that utilizes the automatically discovered correlation patterns to increase the statistical power of AS detection. As such, it represents a novel development for the analysis of allelespecificity.
Model, algorithm, and possible extensions
Unlike tools such as AlleleSeq which mainly focus on the preprocessing steps for the AS analysis (e.g., construction of diploid personal genome), iASeq is developed as a general model working downstream of the preprocessing pipelines. The input data for iASeq are the read counts in the format shown in Figure 1a. With this design, iASeq can be easily coupled with different data preprocessing protocols. For instance, some investigators may map their reads to a reference genome, while others may map their reads to a diploid personal genome. Both types of investigators can use iASeq to integrate information from multiple datasets once they obtained the allelic read counts.
In iASeq, we used an EM algorithm to find the posterior mode of parameters and carried out statistical inference accordingly. In principle, one may also use a full Bayesian approach and Markov Chain Monte Carlo (MCMC) to perform the posterior inference. However, since MCMC usually takes much longer to run for a big dataset and it is not easy for users to monitor convergence, we decided to use the posterior mode and EMbased approach in our implementation. For analyzing the GM12878 data with 94,519 SNPs, iASeq took 5 hours to run the EM algorithm to fit a single model with K=1 on a machine with 2.7 GHz CPU and 4Gb RAM. To fit a single model with K=10 on the same machine, the EM took 16 hours. Running the EM for all 10 K s between 1 and 10 on a single core took 4.6 days. However, when we run these 10 jobs in parallel on 10 cluster nodes, we were able to select the best model within 1 day. Therefore, running the algorithm on a single machine is a little timeconsuming, but the computation time can be reduced by parallelization. Also, our analysis of GM12878 data indicates that the optimal K in that real data was 2. For a K not extremely large, even if running the full BIC selection on a single machine takes some time, it usually requires less than a week, which is acceptable compared to the time devoted to preparing samples and generating data.
In principle, the statistical model developed in iASeq may also be applied to analyze other types of AS events, such as ASE and ASM. In the future, we plan to improve the model by incorporating information from the spatial correlation among closely located SNPs. For example, for the ASE analysis, one may jointly model SNPs from the same gene, similar to [33].
Implications on future studies
The analysis of AS events using the highthroughput sequencing data frequently faces the problem of low statistical power due to the limited amount of information available at heterozygote SNPs. One way to increase the power is to increase the sequencing depth for one data type (e.g., MYC ChIPseq). An alternative approach is to spend the same amount of money to generate data for multiple different but related data types (e.g., ChIPseq for MYC, H3K4me1, H3K4me3, etc.), each with a lower coverage. One can then integrate the multiple datasets to increase the statistical power of allelespecificity analysis. The merit of the second approach is that one can collect multiple different types of information which might be useful for other purposes (e.g., in addition to studying MYC binding using MYC ChIPseq, one may couple H3K4me1 ChIPseq data with DNA motif information to locate active enhancers and predict binding sites of other TFs in the genome). If the second approach is used in the study design, then iASeq will offer a flexible, powerful and scalable framework for better analyzing the AS events in the data. As ChIPseq data continue to grow rapidly, this integrative approach will allow us to use the data more efficiently to characterize the allelespecificity.
Conclusions
In summary, we have proposed a Bayesian hierarchical mixture model iASeq to integrate multiple ChIPseq datasets for analyzing allelespecificity. The primary goal of iASeq is to increase the statistical power of AS detection, and it does so by taking the advantage of correlations among datasets. Since the correlation structure may not be known before the data analysis, iASeq learns it from the data automatically. Application of iASeq to the ENCODE GM12878 data shows that allelic imbalance of most analyzed TFs and HMs have strong preference to be skewed toward the same direction. Analysis of both the simulated and real data show the effectiveness of iASeq to improve detection of allelespecificity compared to singledataset based methods.
Notes
Abbreviations
 AS:

allelespecific
 ASB:

allelespecific binding, including both allelespecific TF binding and allelespecific histone modifications
 ASE:

allelespecific expression
 ASM:

allelespecific DNA methylation
 AUC:

area under receiver operating characteristic curve
 EM:

ExpectationMaximization algorithm
 FDR:

false discovery rate
 HM:

histone modification
 NS:

not allelespecific
 ROC:

receiver operating characteristic curve
 SN:

skewed to the nonreference allele
 SR:

skewed to the reference allele
 TF:

transcription factor.
Declarations
Acknowledgements
This work was supported by the National Institute of Health grant R01HG006841 to HJ, the Strategic Priority Research Program of the Chinese Academy of Sciences, Grant No. XDA01010305 and Hundred Talents Program of the Chinese Academy of Sciences to QW. The authors would like to acknowledge Dr. Joel Rozowsky and Dr. Mark Gerstein for providing AlleleSeq data on GM12878 for the method comparisons.
Authors’ Affiliations
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