MetaDiff: differential isoform expression analysis using random-effects meta-regression
© Jia et al. 2015
Received: 21 May 2014
Accepted: 20 May 2015
Published: 2 July 2015
RNA sequencing (RNA-Seq) allows an unbiased survey of the entire transcriptome in a high-throughput manner. A major application of RNA-Seq is to detect differential isoform expression across experimental conditions, which is of great biological interest due to its direct relevance to protein function and disease pathogenesis. Detection of differential isoform expression is challenging because of uncertainty in isoform expression estimation owing to ambiguous reads and variability in precision of the estimates across samples. It is desirable to have a method that can account for these issues and is flexible enough to allow adjustment for covariates.
In this paper, we present MetaDiff, a random-effects meta-regression model that naturally fits for the above purposes. Through extensive simulations and analysis of an RNA-Seq dataset on human heart failure, we show that the random-effects meta-regression approach is computationally fast, reliable, and can improve the power of differential expression analysis while controlling for false positives due to the effect of covariates or confounding variables. In contrast, several existing methods either fail to control false discovery rate or have reduced power in the presence of covariates or confounding variables. The source code, compiled JAR package and documentation of MetaDiff are freely available at https://github.com/jiach/MetaDiff.
Our results indicate that random-effects meta-regression offers a flexible framework for differential expression analysis of isoforms, particularly when gene expression is influenced by other variables.
The advent of massively parallel sequencing has revolutionized genetics, epigenetics, and transcriptomics studies. RNA sequencing (RNA-Seq) allows an unbiased survey of the entire transcriptome in a high-throughput manner. It has rapidly replaced traditional microarrays as the major platform for transcriptomics studies because it allows for more accurate and a wider range of measurement of gene expression .
A major application of RNA-Seq is to detect differential isoform (i.e., transcript) expression across experimental conditions, which is of great biological interest due to its direct relevance to protein function and disease pathogenesis. Recent evidence suggests that almost all human multi-exon genes have more than one isoform , and different isoforms are often differentially expressed (DE) across tissues, developmental stages, disease conditions, and even across cells from the same tissue [3, 4]. Therefore, detection of DE isoforms is important for understanding complicated biological mechanisms and for mapping disease susceptibility genes.
Detection of DE isoforms using RNA-Seq, however, is challenging because of the uncertainty in isoform expression estimation owing to ambiguous reads and the variability in precision of the estimates across samples. Popular analysis methods such as DESeq , DESeq2 , and EdgeR  expect read counts as input. When using these programs, one would have to estimate the number of fragments originating from each isoform using other programs [8–10] and then analyze the estimated counts as if they were directly observed. Since the estimation of isoform expression may be biased systematically, failing to account for the variability in estimation precision can result in increased false discoveries. Other programs such as Cuffdiff , BitSeq , and EBSeq  can appropriately account for uncertainty in isoform expression estimates. However, these programs cannot be used to adjust for covariates and confounders that may affect gene expression. Recent studies have shown that gene expression can change with environment and age in multiple tissues [14, 15]; for example, Glass et al.  found that ~10-15 % of the genes in the human genome are affected by age in skin, adipose, blood, and brain. In this paper, we refer a covariate as an independent variable that correlates with gene expression but not with the phenotype of interest (e.g., disease status, or treatment group), while a confounder as one that correlates with both gene expression and the phenotype.
A method for detecting DE isoforms ideally should be able to: 1) account for isoform expression estimation uncertainty, 2) account for variation in the precision of isoform expression estimates across biological replicates, and 3) allow adjustment for covariates and confounding factors. Recently, Turro et al.  developed MMDIFF, a Bayesian random-effects method that meets these purposes. However, it is difficult to directly compare MMDIFF with other alternative methods that are frequentist in nature and allow assessment of statistical significance through p-values.
Through extensive simulations and the analysis of an RNA-Seq dataset on human heart failure, we show that MetaDiff is computationally fast, reliable, and can easily incorporate covariates/confounders. In summary, random-effects meta-regression offers a flexible framework for differential expression analysis of isoforms, particularly when gene expression is influenced by other variables.
We evaluated the performance of random-effects meta-regression on both simulated and real RNA-Seq data and compared it with five existing algorithms, including Cuffdiff, DESeq, DESeq2, EdgeR and EBSeq. To make a fair comparison, all programs used FPKMs estimated from Cufflinks, one of the most popular programs for isoform expression estimation. Since DESeq, DESeq2, EdgeR and EBSeq can only take counts as input, we converted the estimated FPKMs into counts for these programs. EBSeq does not explicitly model estimation uncertainty for each isoform, but rather divides isoforms into different groups according to the number of isoforms in a gene. In our analysis, we used the default option in EBSeq that divides the isoforms into three groups. A transcript was declared to be DE if its FDR-adjusted p-value was less than the nominal FDR level (for Cufflinks, DESeq, DESeq2, and EdgeR) or if its posterior probability of DE was greater than one – nominal FDR level (for EBSeq). We did not include MMDIFF in the comparison because it can only take isoform expression estimates obtained from MMSEQ . We note that DESeq, DESeq2, and EdgeR are not designed for differential expression analysis of isoforms. However, given the popularity of these methods, it might be tempting for an end user to identify DE isoforms using FPKM converted counts. Therefore, we included DESeq, DESeq2, and EdgeR in our comparisons.
Overview of random-effects meta-regression
Here i is an index for subject (i = 1, …, n), u i represents the estimation uncertainty for log(Y i ), and e i is random error. To test the null hypothesis of no differential expression between groups, i.e., H0 : β 1 = 0, we use two statistical tests: t-test and Bartlett corrected likelihood-ratio test (BcLR) . Details of the random-effects meta-regression model are described in Methods.
RNA-Seq data simulation
We conducted simulations to evaluate the performance of random-effects meta-regression and compared it with other state-of-the-art algorithms for differential expression analysis. To simulate a realistic dataset, we used Flux Simulator  to generate paired-end RNA-Seq data by modeling RNA-Seq experiments in silico. The human genome sequence (hg19, NCBI build 37) was downloaded from UCSC together with the coordinates of all isoforms in the RefGene table. Flux Simulator assigns an abundance value to each isoform following a mixed power/exponential law. Additionally, it simulates common sources of systematic bias in the abundance and distribution of reads by in silico library preparation and sequencing. We simulated 16 cases and 16 controls. To reflect varying sequencing depth across samples, each individual’s total sequencing output was determined by a uniform distribution from 8 million to 12 million reads.
We first simulated the data without any covariate or confounding effect (Scenario I). We designated 30 % of the transcripts to be DE, half up-regulated in cases by 1.25 fold and half down-regulated by 1.25 fold. The remaining 70 % of the transcripts were assumed to be non-DE between the two groups. This setup allowed us to evaluate both false discovery rate (FDR) and power.
Finally, we considered a variation of Scenario II, in which the covariate was correlated with both gene expression and disease status (Scenario III). We introduced confounding by allowing the covariate to have different distributions: uniform(40,85) for the cases and uniform(18,60) for the controls. The rest of the simulation setup was the same as that in Scenario II (Fig. 2).
For each simulated dataset, the RNA-Seq reads were mapped to the human reference genome using Tophat with default options . Isoform-specific gene expression was estimated using Cufflinks . The estimated FPKM was normalized following the procedure described in DESeq . For each expressed transcript, we tested for differential expression between cases and controls using the t-test and the BcLR test implemented in the R metatest package. We also analyzed the data using popular software packages including Cuffdiff (version 2.1.1), DESeq (version 1.20.0), DESeq2 (version 1.16), EdgeR (version 3.10.0), and EBSeq. Since DESeq, DESeq2, EdgeR and EBSeq can only take counts as input, we converted isoform expression to read count using the formula FPKM × N × L/109, where FPKM is the estimated expression level, N is the total number of mapped reads in the sample, and L is the length of the transcript. We note that Cuffdiff and EBSeq cannot adjust for covariates. For each of the three scenarios, we also evaluated the impact of sample size by analyzing a subset of m cases and m controls (m = 4, 8) randomly chosen from the simulated dataset. We note that Cuffdiff uses a sampling-based method for differential analysis and only generates p-values ≥ 5 × 10−5.
Comparison of empirical FDR in simulated data
Comparison of quantile-quantile(QQ) plots in simulated data
Comparison of power in simulated data
Application to the human heart failure RNA-Seq data
We next evaluated the performance of these methods using a dataset from our ongoing study on human heart failure. RNA sequencing using Illumina HiSeq 2000 was performed on left ventricle samples collected from four cases and three controls in the MAGNet study (http://www.med.upenn.edu/magnet). The heart was perfused with cold cardioplegia prior to cardiectomy to arrest contraction and prevent ischemic damage. Left ventricular free-wall tissue was harvested and snap frozen with liquid nitrogen at the time of cardiac surgery from subjects with heart failure undergoing transplantation and from unused donor hearts. This study was approved by the University of Pennsylvania Institutional Review Board and the Cleveland Clinic Institutional Review Board. All participants were 18 years or older and provided written informed consent. The seven selected subjects had a wide range of age distribution (controls: mean = 47, range [32, 57]; cases: mean = 58.5, range [41, 68]). Among the seven subjects, four were male and three were female. Poly-A library preparation and sequencing was performed at the Penn Genome Frontiers Institute’s High-Throughput Sequencing Facility following standard protocols. The average sequencing depth was 43 million 2 × 101 bp paired-end reads. We mapped the reads to the human reference genome using Tophat, and then applied several filtering criteria to ensure that the reads were uniquely mapped with a mapping quality score ≥ 30 and that two reads in a pair were mapped to the same chromosome with expected orientations and distance < 500,000 bp. We estimated isoform-specific gene expression using Cufflinks.
Number of DE transcripts (FDR adjusted p-value < 0.05 or posterior probability of DE > 0.95) detected by each method in the heart failure dataset
We further examined the 41 DE transcripts that were detected by the age-sex adjusted BcLR test but were missed by the other tests, including age-sex adjusted t-test, DESeq, DESeq2 and EdgeR, and unadjusted BcLR, t-test, DESeq, DESeq2 and EdgeR. Several of these transcripts are from genes that have been implicated in heart failure pathogenesis. For example, Schattermann et al.  reported that PDGFA is required for normal murine cardiovascular development. Wang et al.  showed that PLCE1 is upregulated in human hearts during heart failure, which is consistent with elevated expression levels of the DE isoform (NM_001165979) observed in our heart failure samples. Proteins encoded by the SMYD family are shown to be key regulators in skeletal and cardiac muscle development and function . TMEM88 encodes a protein that is crucial for heart development and acts downstream of GATA factors in the pre-cardiac mesoderm. It is worth noting that among the genes for which the 41 DE transcripts originate from, only about half were detected to be DE by gene level analysis using the age-sex adjusted BcLR test. This suggests that if one were to perform gene-only analysis, signals at the isoform level would have been missed.
One of the major applications of RNA-Seq is to identify DE isoforms. In differential expression analysis, it is important to account for the fact that isoform expression levels are estimated rather than observed, that they are estimated with various precision across samples, and that covariates and confounding factors may play a role to influence gene expression. To do so, we have proposed a flexible regression framework, utilizing the well-established random-effects meta-regression approach. Through computer simulations and the analysis of a real RNA-Seq dataset on human heart failure, we demonstrated that the proposed method can improve the power of isoform differential analysis while controlling for false positives due to the effect of covariates or confounding variables. The meta-regression approach we used is computationally efficient and widely available in existing statistical software packages. We have provided a tool and instructions on how to use meta-regression for isoform differential expression analysis with RNA-Seq data.
We have compared the performance of our method and other commonly used methods for differential expression analysis, including Cuffdiff, DESeq, DESeq2, EdgeR, and EBSeq. Both Cuffdiff and EBSeq take into account the estimation uncertainty for isoform expression levels, although EBSeq does not explicitly model the degree of uncertainty. In our simulated data, when no covariate and confounder influenced isoform expression (Scenario I), Cuffdiff had lower power than our method when m = 8 or 16, but better power when sample size was small (m = 4). In contrast, EBSeq had conservative FDR among non-DE transcripts and correspondingly lower power for detecting true DE transcripts. When a covariate or confounder was present, these two methods showed either lower statistical power (in Scenario II) or inflated FDR (in Scenario III) as they are unable to adjust for covariates.
DESeq, DESeq2, and EdgeR are based on negative binomial regression to model read counts from an RNA-Seq experiment, and thus it is natural for these methods to incorporate covariates. To estimate the variance in a negative binomial distribution, EdgeR relates the variance to the mean through a dispersion parameter that is constant across all genes. DESeq, on the other hand, applies a local regression to allow the dispersion parameter to vary from gene to gene. DESeq2 is a successor of DESeq. It uses shrinkage estimation to estimate dispersion parameters and fold change, thus offering improved stability as compared to DESeq. Additionally, DESeq2 allows adjustment of continuous covariates, whereas DESeq can only adjust discrete covariates. In our simulations, although EdgeR could control FDR in the presence of a confounder, it was conservative in all three scenarios. It also had lower power to detect true DE transcripts compared to our method and Cuffdiff in Scenario I. In contrast, DESeq showed inflated FDR for non-DE transcripts regardless the presence of confounder, especially when sample size was small. When a covariate or a confounder was present, DESeq had little power to detect DE transcripts that were correlated with the covariate. DESeq2 showed better performance than DESeq, however, its overall performance was not as satisfactory as BcLR and t-test. We note that EdgeR, DESeq, and DESeq2 cannot take into account the uncertainty in isoform expression estimation, which may lead to biased testing results.
The uncertainty in an isoform FPKM estimate can be quantified as a standard error, which can be calculated in Cufflinks. In differential expression analysis, the FPKM value is usually log-transformed, and we approximate the variance of log(FPKM) using the delta method, which is also used in Cuffdiff 1.0 . However, this approximation can be poor and lead to false positive results when the variance of FPKM value is large compared to its magnitude. Hence we filtered out transcripts with large CVs in meta-regression. To avoid using delta method to approximate the variance of log(FPKM), one could use MMSEQ estimated isoform expression because MMSEQ directly gives the variance estimate of log(FPKM). Since DE transcripts that were influenced by a covariate or a confounder tended to have a higher CV than those not influenced by covariates/confounders, the true DE transcripts that were filtered out would have a higher proportion in the former. This is confirmed in Additional file 2: Table S1. If we had not applied the filter, the power of our proposed approach would have been even higher, but at the expense of inflated false positive results. We are in search of alternative filtering criteria to minimize the number of true DE transcripts being filtered out.
We note that filtering is a commonly used strategy to eliminate potential false positive signals in genomic data analysis. Other methods have also employed filtering. For example, Cuffdiff gives NOTEST if the number of reads is smaller than some internally defined threshold. In EBSeq, those whose 75th quantile of normalized counts is less than 10 are also filtered because lowly expressed genes are more likely to be affected by noises. In our experience, transcripts with a high CV typically have low expression levels because lowly expressed transcripts generally have smaller number of reads mapped to them and this would lead to relatively higher estimation uncertainty and thus high variability of expression estimates among samples. We evaluated the impact of using various CV thresholds on filtering by examining empirical FDRs and QQ plots. Through simulations and sensitivity analysis, we chose a cutoff of 0.4, which gives satisfactory FDRs and QQ plots for all simulation settings we considered. We note that CV filtering could also help eliminate false positive results in the other methods evaluated in this paper, although these methods didn’t choose to use CV as a filter.
We note that Cuffdiff (version 2.1.1) implemented a sampling-based method for differential analysis instead of estimating the standard error of log-transformed FPKM, and this approach led to slightly better results as compared to the delta method approximation. However, the resolution of its p-value is limited by the number of samplings, and the current implementation of the program generates p-values only up to 5 × 10−5. When a more stringent p-value threshold is desired, the computational time will be significantly increased with an increased number of samplings.
Throughout the paper, we have used FPKMs estimated from Cufflinks as input for various evaluations. We note that meta-regression only requires estimates of isoform expression and the corresponding estimation uncertainty, but it is not tied to any particular estimation method. However, to evaluate the potential impact of isoform expression estimation method on our results, we compared the estimated isoform expression levels using three different software packages, including Cufflinks, RSEM, and MMSEQ. Our evaluation indicates that these three software packages gave highly similar expression estimates (Additional file 2). The choice of expression estimation method has little impact on our results, and this was confirmed by comparing MetaDiff with Cufflinks and MMSEQ estimated isoform expressions (Additional file 2).
In this paper, we explained the equivalence between a random-effects model that accounts for estimation uncertainty in differential expression analysis and the random-effects meta-regression. Meta-regression has been well studied in statistics and epidemiology literatures [25–27], and is easy to implement using standard software. Both RNA-Seq analysis and meta-analysis face the same problem of small sample size. The BcLR test uses a correction factor to modify the standard LR test for small sample sizes. Huizenga et al.  compared several testing procedures for meta-regression and showed that the BcLR test and t-test are the two best options. In our simulation study, we found that the BcLR test outperformed t-test with less conservative FDR and more power to detect DE transcripts.
Meta-regression is similar in nature to MMDIFF , a recently developed Bayesian random-effects model for isoform differential expression analysis. We did not include MMDIFF in our evaluations because MMDIFF can only take MMSEQ  expression estimates as input. However, our additional analyses indicate that results from MMDIFF and meta-regression are highly concordant. Since meta-regression is frequentist-based and yields p-values, we believe it offers a useful alternative to MMDIFF.
In summary, we have proposed MetaDiff, a flexible regression framework for isoform differential expression analysis that can take into account isoform expression estimation uncertainty and variation across biological replicates, and allow for covariate adjustment. Our method can effectively control for false positives due to confounding and increase the power to detect true DE transcripts. MetaDiff can be freely downloaded from https://github.com/jiach/MetaDiff. This regression framework is flexible and can be readily extended to other study designs, e.g., paired data and time course data.
When the FPKM values were 0, we add 0.0001 in order to avoid negative infinity in the log transformation.
Random-effects meta-regression for testing differential expression
Here u i represents the estimation uncertainty for log(Y i ), and e i is random error. For this random-effects model, we assume: 1) u i ~ N(0, σ 1i 2 ), 2) e i ~ N(0, τ 2), 3) u i and e i are uncorrelated, i.e., Cov(u i , e i = 0), and 4) the n observations are independent. We note that equation (2) is identical to the random-effects meta-regression model that is widely used in meta-analysis . Because of this, we can readily carry out statistical inference using standard statistical software, such as R (http://www.r-project.org) and Stata (Stata Corp, College Station, TX). In our analysis, we used the metatest package in R .
RNA-Seq data have been deposited in the Gene Expression Omnibus (GEO) database (accession number GSE57344).
This research was supported by the National Institutes of Health [R01GM097505, R01GM108600, R01HG006465 to ML; R01HG004517 to CL; R01HL088577 and R01HL105993 to TPC; R01HL105993 to KBM; R01HL105993 and R01HL103931 to WHWT]. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health. This research was also supported by the Kaufman Center for Heart Failure at the Cleveland Clinic.
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.
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