# MATS: a Bayesian framework for flexible detection of differential alternative splicing from RNA-Seq data

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**ABSTRACT:**Background Alternative Splicing (AS) as a post-transcription regulation mechanism is an important application of RNA-seq studies in eukaryotes. A number of software and computational methods have been developed for detecting AS. Most of the methods, however, are designed and tested on animal data, such as human and mouse. Plants genes differ from those of animals in many ways, e.g., the average intron size and preferred AS types. These differences may require different computational approaches and raise questions about their effectiveness on plant data. The goal of this paper is to benchmark existing computational differential splicing (or transcription) detection methods so that biologists can choose the most suitable tools to accomplish their goals.ResultsThis study compares the eight popular public available software packages for differential splicing analysis using both simulated and real Arabidopsis thaliana RNA-seq data. All software are freely available. The study examines the effect of varying AS ratio, read depth, dispersion pattern, AS types, sample sizes and the influence of annotation. Using a real data, the study looks at the consistences between the packages and verifies a subset of the detected AS events using PCR studies.Conclusions No single method performs the best in all situations. The accuracy of annotation has a major impact on which method should be chosen for AS analysis. DEXSeq performs well in the simulated data when the AS signal is relative strong and annotation is accurate. Cufflinks achieve a better tradeoff between precision and recall and turns out to be the best one when incomplete annotation is provided. Some methods perform inconsistently for different AS types. Complex AS events that combine several simple AS events impose problems for most methods, especially for MATS. MATS stands out in the analysis of real RNA-seq data when all the AS events being evaluated are simple AS events.BMC Bioinformatics 12/2014; 15(1):364. · 2.67 Impact Factor - SourceAvailable from: sciencedirect.com
##### Article: PrimerSeq: Design and Visualization of RT-PCR Primers for Alternative Splicing Using RNA-seq Data

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**ABSTRACT:**The vast majority of multi-exon genes in higher eukaryotes are alternatively spliced and changes in alternative splicing (AS) can impact gene function or cause disease. High-throughput RNA sequencing (RNA-seq) has become a powerful technology for transcriptome-wide analysis of AS, but RT-PCR still remains the gold-standard approach for quantifying and validating exon splicing levels. We have developed PrimerSeq, a user-friendly software for systematic design and visualization of RT-PCR primers using RNA-seq data. PrimerSeq incorporates user-provided transcriptome profiles (i.e., RNA-seq data) in the design process, and is particularly useful for large-scale quantitative analysis of AS events discovered from RNA-seq experiments. PrimerSeq features a graphical user interface (GUI) that displays the RNA-seq data juxtaposed with the expected RT-PCR results. To enable primer design and visualization on user-provided RNA-seq data and transcript annotations, we have developed PrimerSeq as a stand-alone software that runs on local computers. PrimerSeq is freely available for Windows and Mac OS X along with source code at http://primerseq.sourceforge.net/. With the growing popularity of RNA-seq for transcriptome studies, we expect PrimerSeq to help bridge the gap between high-throughput RNA-seq discovery of AS events and molecular analysis of candidate events by RT-PCR.Genomics Proteomics & Bioinformatics 01/2014; - SourceAvailable from: Kristoffer Vitting-Seerup[Show abstract] [Hide abstract]

**ABSTRACT:**RNA-seq data is currently underutilized, in part because it is difficult to predict the functional impact of alternate transcription events. Recent software improvements in full-length transcript deconvolution prompted us to develop spliceR, an R package for classification of alternative splicing and prediction of coding potential. spliceR uses the full-length transcript output from RNA-seq assemblers to detect single or multiple exon skipping, alternative donor and acceptor sites, intron retention, alternative first or last exon usage, and mutually exclusive exon events. For each of these events spliceR also annotates the genomic coordinates of the differentially spliced elements, facilitating downstream sequence analysis. For each transcript isoform fraction values are calculated to identify transcript switching between conditions. Lastly, spliceR predicts the coding potential, as well as the potential nonsense mediated decay (NMD) sensitivity of each transcript. spliceR is an easy-to-use tool that extends the usability of RNA-seq and assembly technologies by allowing greater depth of annotation of RNA-seq data. spliceR is implemented as an R package and is freely available from the Bioconductor repository (http://www.bioconductor.org/packages/2.13/bioc/html/spliceR.html).BMC Bioinformatics 03/2014; 15(1):81. · 2.67 Impact Factor

Page 1

MATS: a Bayesian framework for flexible

detection of differential alternative splicing

from RNA-Seq data

Shihao Shen1, Juw Won Park2, Jian Huang3, Kimberly A. Dittmar4, Zhi-xiang Lu2,

Qing Zhou5, Russ P. Carstens4,6and Yi Xing2,7,*

1Department of Biostatistics,2Department of Internal Medicine,3Department of Statistics and Actuarial Science,

University of Iowa, Iowa City, IA 52242,4Renal Division, Department of Medicine, University of Pennsylvania,

School of Medicine, Philadelphia, PA 19104,5Department of Statistics, University of California, Los Angeles, CA

90095, USA,6Department of Genetics, University of Pennsylvania School of Medicine, Philadelphia, PA 19104

and7Department of Biomedical Engineering, University of Iowa, Iowa City, IA 52242

Received August 6, 2011; Revised December 10, 2011; Accepted December 15, 2011

ABSTRACT

Ultra-deep RNA sequencing has become a powerful

approach for genome-wide analysis of pre-mRNA

alternative splicing. We develop MATS (multivariate

analysis of transcript splicing), a Bayesian statistical

framework for flexible hypothesis testing of differ-

ential alternative splicing patterns on RNA-Seq data.

MATS uses a multivariate uniform prior to model

the between-sample correlation in exon splicing

patterns, and a Markov chain Monte Carlo (MCMC)

method coupled with a simulation-based adaptive

sampling procedure to calculate the P-value and

false discovery rate (FDR) of differential alternative

splicing. Importantly, the MATS approach is applic-

able to almost any type of null hypotheses of

interest, providing the flexibility to identify differen-

tial alternative splicing events that match a given

user-defined pattern. We evaluated the perform-

ance of MATS using simulated and real RNA-Seq

data sets. In the RNA-Seq analysis of alternative

splicing events regulated by the epithelial-specific

splicing factor ESRP1, we obtained a high RT–PCR

validation rate of 86% for differential exon skipping

events with a MATS FDR of <10%. Additionally,

over the full list of RT–PCR tested exons, the

MATS FDR estimates matched well with the experi-

mental validation rate. Our results demonstrate

that MATS is an effective and flexible approach for

detectingdifferential alternative

RNA-Seq data.

splicingfrom

INTRODUCTION

Alternative splicing plays critical roles in regulating gene

function and activity in higher eukaryotes (1). By alterna-

tive selection of exons and splice sites during splicing, a

single gene locus can produce multiple mRNA and protein

isoforms with divergent structural and functional pro-

perties (2). Over 90% of multi-exon human genes are

alternatively spliced and many genes undergo changes in

alternative splicing during development, cell differenti-

ation and disease (3–6). Changes in alternative splicing

patterns can be manifested as all-or-none switches between

distinct mRNA isoforms, but more frequently as shifts in

the relative abundance of multiple mRNA isoforms of a

gene. In some disease genes, a slight shift (by as few as

several percent) in the relative isoform proportions can

trigger disease pathogenesis (7,8). Due to the prevalent

role of alternative splicing in gene regulation and disease,

there is a pressing need for genomic tools that can accur-

ately quantify isoform ratios and reliably detect changes in

isoform ratios (i.e. differential alternative splicing) among

different biological conditions.

Direct sequencing of full-length mRNAs and mRNA

fragments has been a popular and effective approach for

discovery and quantification of alternative splicing events

(3,4,9). Since mRNAs represent the end products of

splicing, by aligning mRNA sequences to the genome

one can delineate exon–intron structures and identify al-

ternative splicing events. If sequencing reaches the suffi-

cient depth, such that the relative abundance of distinct

mRNA isoforms can be confidently estimated by the

numbers of RNA sequences matching to specific isoforms,

we can compare the mRNA sequence counts across bio-

logical conditions to identify differential alternative

*To whom correspondence should be addressed. Tel: +1 319 384 3099; Fax: +1 319 384 3150; Email: yi-xing@uiowa.edu

Published online 20 January 2012Nucleic Acids Research, 2012, Vol. 40, No. 8e61

doi:10.1093/nar/gkr1291

? The Author(s) 2012. Published by Oxford University Press.

This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/

by-nc/3.0), which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

Page 2

splicing events. This approach was first adopted for the

discovery of tissue-specific exons from expressed sequence

tags (ESTs) (10). Xu et al. categorized the ESTs of human

genes according to their tissue origins. The exon inclusion

level of an alternatively spliced cassette exon in any given

tissue was estimated from the counts of ESTs mapped

uniquely to the exon inclusion or skipping exon-exon junc-

tions (10) (for a formal definition, see ‘Materials and

Methods’ section). Using a Bayesian approach, Xu et al.

compared the EST counts across different tissues to detect

exons with tissue-specific shifts in exon inclusion levels.

Specifically, the prior distribution of an exon’s exon inclu-

sion levels in two tissues was modeled as two independent

uniform (0, 1) distributions. The EST read count of the

exon inclusion isoform in each tissue was assumed to

follow a binomial distribution. A Bayesian posterior

probability was calculated to assess whether the exon in-

clusion levels of an exon differed between two tissues.

Similar approacheswere

cancer-specific alternative splicing events (11,12). These

studies pioneered the use of RNA sequence count data

for quantitative splicing analysis. However, due to the

low throughput of EST sequencing, EST-based analysis

of differential alternative splicing has limited capacity

and high noise (13).

Recently, with the advent of the high-throughput

RNA sequencing technology (RNA-Seq), it has become

feasible to generate hundreds of millions of short

RNA-Seq reads on any RNA sample of interest (14). This

technology enables genome-wide quantitative analyses of

RNA alternative splicing (3,4). Pan and colleagues

demonstrated that if the RNA-Seq coverage of an alterna-

tively spliced cassette exon reaches at least 20 reads for one

of its exon–exon junctions, the exon inclusion levels

estimated by RNA-Seq strongly correlate with splicing

microarray measurements (4). Other studies comparing

RNA-Seq data to RT–PCR data reached similar conclu-

sions (15–17). Thus, by analyzing and comparing deep

RNA-Seq data from different biological conditions, one

can identify exons with changes in exon inclusion levels

onagenomescale.FromtheRNAsequencecountdata,dif-

ferentialalternativesplicingeventsarecommonlyidentified

by testing the equality of transcript isoform ratios between

samples (11,15,16,18–20). Various methods have been used

toassessthestatisticalsignificanceofsuchdifferentialalter-

native splicing events, including Fisher exact test of

isoform-specific read counts (15,19,20), and Bayesian

approachesthatmodelreadcountsassampledfromaprob-

abilistic mixture of distinct isoforms (11,16,21).

In this article, we introduce MATS (multivariate

analysis of transcript splicing), a Bayesian statistical

framework for flexible hypothesis testing of differential

alternativesplicingpatterns

Compared to previous computational methods for detect-

ing differential alternative splicing events from RNA

sequence count data, MATS has several novel features.

First and most importantly, MATS offers the flexibility

to identify differential alternative splicing events that

match a given user-defined pattern. For example, MATS

can calculate the statistical significance that the absolute

difference in the exon inclusion levels of an exon between

laterused toidentify

onRNA-Seqdata.

two conditions exceeds a given threshold (e.g. 10%). This

allows biologists to identify alternative splicing changes

that reach any specified magnitude. MATS can also be

used to detect exons with the extreme ‘switch-like’ differ-

ential alternative splicing pattern, i.e. exons predominant-

ly included in the transcripts in one condition but

predominantly skipped in

switch-like pattern is of considerable biological interest,

because it is a strong indicator of functional alternative

splicing events (3,22). Second, MATS uses a multivariate

uniform distribution as the joint prior for the exon inclu-

sion levels in two conditions. Compared to the independ-

ent uniform priors commonly used by previous methods,

the multivariate uniform prior is more general and better

captures the genome-wide similarity in exon splicing

patterns between biological conditions. Of note, this

prior distribution is motivated by the observation that

between any two conditions there is generally an overall

positive correlation in exon splicing patterns, and only a

small percentage of alternatively spliced exons undergo

differentialsplicing(see‘Results’

MATS employs a Markov chain Monte Carlo (MCMC)

method coupledwitha

sampling procedure to calculate the P-value and false dis-

covery rate (FDR) of differential alternative splicing, by

comparing the posterior probability of the observed data

to a set of posterior probabilities simulated from the null

hypothesis. This approach is applicable to almost any type

of null hypotheses of interest. To evaluate the perform-

ance of MATS, we analyzed a deep RNA-Seq data set

with 256 million reads on a human breast cancer cell line

(MDA-MB-231) with ectopic expression of the epithelial-

specific splicing factor ESRP1 or an empty vector (EV)

control. In this experimental system, the splicing factor

ESRP1 induces large-scale changes in alternative splicing

towards an epithelial-specific splicing signature (23).

Based on the MATS result, we selected 164 exons that

covered a broad range of FDR values for RT–PCR valid-

ation.ForexonswithaMATSFDRof<10%,weobtained

ahigh overall RT–PCR

demonstrating that MATS can reliably detect differential

alternative splicing events. Additionally, over the full list

of RT–PCR tested exons, we observed a progressive

decrease in the RT–PCR validation rate with increasing

FDR values, suggesting MATS can yield experimentally

meaningful FDR estimates to help biologists interpret

RNA-Seq predictions and design follow-up experiments.

anothercondition.This

section).Finally,

simulation-basedadaptive

validationrate of86%,

MATERIALS AND METHODS

Overview of MATS

MATS is a Bayesian statistical framework to detect dif-

ferential alternative splicing events from RNA-Seq data.

For every alternatively spliced cassette exon, MATS

assesses the statistical significance of differential alterna-

tive splicing based on a user-defined hypothesis. The

default analysis of MATS is to test whether the difference

in the exon inclusion levels between two samples exceeds a

given user-defined threshold (e.g. 10%). Compared to

existing RNA-Seq analysis methods that test the equality

e61Nucleic Acids Research, 2012,Vol. 40,No. 8PAGE 2 OF 13

Page 3

of exon inclusion levels between samples, the MATS test

of splicing difference has three advantages. First, it

provides a rigorous statistical framework for biologists

to identify alternative splicing changes that reach any

specified magnitude. Second, it improves robustness

against random sampling noise in the RNA-Seq data,

which could cause a minor shift in the estimated isoform

ratio between samples. For exons with high RNA-Seq

read counts such as those from highly expressed genes,

such random noise might introduce false positives to a

test of equality on exon inclusion levels. Third, the

flexible hypothesis formulation also allows testing of

other types of differential alternative splicing behavior

such as the ‘switch-like’ pattern, in which an exon is pre-

dominantly included in the transcripts in one condition

but predominantly skipped in another condition.

The major steps of MATS are illustrated schematically

in Figure 1. First, for each exon MATS uses the counts of

RNA-Seq reads mapped to the exon-exon junctions of its

inclusion or skipping isoform to estimate the exon inclu-

sion levels in two samples (Figure 1A). Second, the exon

inclusion levels of all alternatively spliced cassette exons

are used to construct a multivariate uniform prior that

models the overall similarity in alternative splicing profiles

between the two samples (Figure 1B). Third, based on the

multivariate uniform prior and a binomial likelihood

model for the RNA-Seq read counts of the exon inclu-

sion/skipping isoforms, MATS uses a MCMC method

to calculate the Bayesian posterior probability for

splicing difference. Under the default setting, MATS cal-

culates the posterior probability that the change in the

exon inclusion level of a given exon exceeds a given

user-defined threshold (e.g. 10%; Figure 1C). Finally,

MATS calculates a P-value for each exon by comparing

the observed posterior probability (from Step C) with a set

of simulated posterior probabilities from the null hypoth-

esis (Figure 1D). These P-values are then transformed to

FDR values by the Benjamini–Hochberg FDR method

(24). Below we describe the details of the MATS

algorithm.

Estimating exon inclusion levels

We define the exon inclusion level ( ) of an alternatively

spliced cassette exon as the percentage of ‘exon inclusion’

transcripts that splice from its upstream exon into the

cassette exon then into its downstream exon among all

such ‘exon inclusion’ transcripts plus ‘exon skipping’ tran-

scripts that splice from its upstream exon directly into its

downstream exon (16,17,25). In a RNA-Seq study, for

each exon in a given sample we count the number of

RNA-Seq reads uniquely mapped to its upstream, down-

stream or skipping exon–exon junctions (Figure 1A). The

upstream junction count (UJC) and the downstream

junction count (DJC) reflect the abundance of the exon

inclusion isoform, while the skipping junction count (SJC)

reflects the abundance of the exon skipping isoform. Let I

and S represent the counts of exon inclusion and skipping

isoforms respectively. Assuming that the read counts

follow a binomial distribution, the maximum likelihood

estimate (MLE) of the exon inclusion level ( ) of an

exon in a given sample can be calculated as:

^ ¼ I=ðI+SÞ ¼

ðUJC+DJCÞ=2

ðUJC+DJCÞ=2+SJC

Calculating the Bayesian posterior probability for

differential alternative splicing

To compare alternative splicing patterns between two

samples, for each exon we define 1 and 2 as its exon

inclusion levels in sample 1 and 2. Under the default

setting, MATS tests the hypothesis that the difference in

the exon inclusion levels of a given exon between sample 1

and 2 is above a user-defined cutoff c, i.e. j 1? 2j > c.

The cutoff c is a user-defined parameter that represents the

extent of splicing change one wishes to identify. For

example, if a researcher is interested in identifying exons

with at least 10% change in exon inclusion levels, the

cutoff c should be set as 10%. The values of 1and 2

under the null hypothesis (H0) and the alternative hypo-

thesis (H1) of this test are illustrated graphically in

Figure 1. Basic steps of MATS. (A) For each exon MATS uses the

counts of RNA-Seq reads mapped to the exon–exon junctions of its

inclusion or skipping isoform to estimate the exon inclusion levels in

two samples. (B) The exon inclusion levels of all alternatively spliced

cassette exons are used to construct a multivariate uniform prior that

models the overall similarity in alternative splicing profiles between the

two samples. (C) Based on the multivariate uniform prior and a

binomial likelihood model for the RNA-Seq read counts of the exon

inclusion/skipping isoforms, MATS uses a Markov chain Monte Carlo

(MCMC) method to calculate the Bayesian posterior probability for

splicing difference. (D) MATS calculates a P-value for each exon by

comparing the observed posterior probability with a set of simulated

posterior probabilities from the null hypothesis, followed by adjustment

for multiple testing to obtain the FDR value.

PAGE 3 OF 13Nucleic AcidsResearch, 2012, Vol.40,No. 8e61

Page 4

Figure 2A. In this section, we describe how MATS calcu-

lates the posterior probability of j 1? 2j > c from the

RNA-Seq counts, i.e. Pðj 1? 2j > cjDataÞ. The P-value

and FDR calculation is described in the next section.

To calculate the posterior probability of j 1? 2j > c,

we need to define the prior probability and the likelihood

model. In MATS, the joint prior distribution of 1and 2

is modeled as a multivariate uniform distribution (Figure

1B), with marginal distributions as uniform (0, 1) and cor-

relation ? ? uniform (0, 1):

Prior :ð 1, 2Þ ? MultiVarUniform 0,1,cor ¼

? ? Uniformð0,1Þ

The multivariate uniform distribution was obtained

by applying cumulative standard normal distribution

functions to a random vector that follows a multivariate

normaldistribution.Specifically,

?ðZ2ÞÞ, where Z1 and Z2 are standard normal random

variables Nð0,1Þ with correlation ? and ? is the cumulative

distribution function of the standard normal distribution.

The obtained multivariate uniform distribution is equiva-

lent to a bivariate distribution with uniform marginals.

We note that our choice of prior distribution in MATS

differs from previous methods which model the priors of

1 and 2 as two independent uniform distributions

(11,16). This multivariate uniform prior distribution of

1and 2is motivated by the observation that between

any two biological conditions, there is generally an overall

similarity in the genome-wide alternative splicing profiles,

and only a small percentage of alternatively spliced exons

undergo differential splicing. Indeed, our analysis of

several RNA-Seq data sets suggests that this multivariate

uniform prior provides a good fit with empirical data

(see ‘Results’ section). In contrast, the commonly used

independent uniform prior distributions assume that the

splicing activities of the same exon in two different

samples are independent, even if these two samples have

1

?

?

1

????

ð 1, 2Þ ¼ ð?ðZ1Þ,

the identical cell type and tissue origin. This lacks bio-

logical justification and fits empirical data poorly.

In each sample, the exon inclusion isoform count I

follows a binomial distribution with n ¼ I+S and p ¼ ,

where S is the skipping isoform count.

Assume we have a total of N alternatively spliced

cassette exons, for each exon i ¼ 1,:::,N, we denote:

i1, i2: exon inclusion levels of exon i in sample 1 and 2;

Ii1,Ii2: counts of the exon inclusion isoform of exon i in

sample 1 and 2;

Si1,Si2: counts of the exon skipping isoform of exon i in

sample 1 and 2.

Likelihood : Ii1j i1 ? Binomial ðni1¼ Ii1+Si1,pi1¼ i1Þ

Ii2j i2 ? Binomial ðni2¼ Ii2+Si2,pi2¼ i2Þ

The posterior probability of differential alternative

splicing for exon i can be calculated as Pi¼ Pðj i1? i2j

> cjIi1,Ii2,Si1,Si2,I½?i?1,I½?i?2,S½?i?1,S½?i?2Þ, where the counts

of the exon inclusion/skipping isoforms of exon i and all

other alternatively spliced cassette exons (indexed by [?i])

are used to infer the parameter ? in the multivariate

uniform prior as well as i1 and i2, and c represents

the user-defined threshold for splicing change.

As this posterior probability cannot be calculated with

an analytic solution in closed-form, we adopt a numeric

solution based on the MCMC method. Specifically, the

posterior probability is calculated by the JAGS program

(Just Another Gibbs Sampler; http://sourceforge.net/

projects/mcmc-jags/). This program estimates the poster-

ior probability Pðj i1? i2j > cjIi1,Ii2,Si1,Si2,I½?i?1,I½?i?2,

S½?i?1,S½?i?2Þ for all exons and the parameter ? of the multi-

variate uniform prior. The parameter ? is a global param-

eter for all exons, which describes the overall correlation

of the exon inclusion levels of all alternatively spliced

exons between two samples. Therefore, for N exons

there are a total of 2N+1 parameters, including 2N par-

ameters for exon inclusion levels in two samples and the

Figure 2. Null hypotheses in MATS. (A) Under the default setting of MATS, the H1 alternative hypothesis is that the difference in the exon

inclusion levels between two samples is above the user-defined cutoff c (the white area). The H0null hypothesis is that the difference is below the

user-defined cutoff c (the gray area). (B) MATS can test the extreme ‘switch-like’ differential alternative splicing pattern with a different hypothesis.

The H1alternative hypothesis is that the exon inclusion level is below a user-defined threshold s in sample 1 and above 1-s in sample 2, or vice versa

(the white area). The H0 null hypothesis is outside the alternative hypothesis region (the gray area).

e61Nucleic Acids Research, 2012,Vol. 40,No. 8PAGE 4 OF 13

Page 5

global parameter ?. To estimate the global parameter ?

along with all the values, the data of all N exons are

used as the input for the MCMC sampler. We burn in the

MCMC sampler for 2000 iterations, followed by another

10000 iterations to calculate the posterior probabilities.

The posterior probability of a given exon i (denoted as

Pobs

i

) is estimated by the fraction of iterations with

j i1? i2j ? c among all 10000 iterations (Figure 1C).

Calculating the P-value and FDR for differential

alternative splicing

We use a simulation-based adaptive sampling procedure

to calculate the P-value and FDR for differential alterna-

tive splicing. In theory, P-value comes from the compari-

son of the observed test statistics with statistics from the

null hypothesis. In MATS, when we test j 1? 2j > c, we

consider the null hypothesis that j 1? 2j ? c. We calcu-

late the P-value of each exon by comparing the posterior

probability of the observed data (Pobs

probabilities simulated from the null hypothesis. For each

exon, we find the maximum likelihood estimate (MLE) of

the constrained 1and 2(denoted as^ c

the binomial distributions with the counts I1,I2,S1and S2

in two conditions,subject

j 1? 2j ? c. Specifically,

ð^ c

i

) to a set of posterior

1and^ c

2) from

to theconstraint that

1,^ c

2Þ ¼ argmax

j 1? 2j?cðI1log 1+S1logð1 ? 1Þ+I2log 2

+S2logð1 ? 2ÞÞ

limited-memoryBroyden–Fletcher–Goldfarb–

Shanno box-constraints (L-BFGS-B) algorithm is used

to search for the constrained MLE (26,27). Then we

simulate RNA-Seq count data from the constrained

MLE of^ c

2, and calculate the posterior probability

of j 1? 2j > c given the simulated data. The P-value of

each exon is calculated by comparing the posterior prob-

ability of differential splicing based on the observed data

to a set of simulated posterior probabilities. The details of

this calculation for a given exon i are summarized below:

The

1and^ c

(1) Retrieve the estimated global parameter ^ ? from the

MCMC calculation of posterior probabilities of all

alternatively spliced exons. The value of ^ ? is fixed in

the P-value calculation.

(2) For exon i, retrieve the observed posterior prob-

abilityfrom theMCMC

Pðj i1? i2j > cjIi1,Ii2,Si1,Si2,I½?i?1,I½?i?2,S½?i?1,S½?i?2Þ.

(3) For exon i, simulate M posterior probabilities from

the constrained MLE of^ c

i) Simulate data

calculationPobs

i

¼

i1and^ c

i2. For j ¼ 1,:::,M:

Ii1jj^ c

Si1j¼ ni1? Ii1j

Ii2jj^ c

Si2j¼ ni2? Ii2j

ii) Calculate the posterior probability from the simu-

lated data Ii1j,Ii2j,Si1j,Si2jusing the MCMC method

as Psim

ij

¼ Pðj i1j? i2jj > cjIi1j,Ii2j,Si1j, Si2j,^ ?Þ.

i1? Binomial ðni1¼ Ii1+Si1,pi1¼^ c

i1Þ;

i2? Binomial ðni2¼ Ii2+Si2,pi2¼^ c

i2Þ;

iii) Calculate the P-value for exon i by comparing

Pobs

i

with thesimulated

IðPobs

For each exon, the number of M is determined by an

adaptive sampling procedure (see below).

The number of simulations (M) in calculating the

simulated posterior probabilities is determined by an

adaptive sampling procedure. Initially, we aim to reach

a P-value precision of 0.01 by setting M=100. One

hundred simulated posterior probabilities are calculated

for each exon, and the exon’s P-value is generated by

comparing the observed posterior probability to the

simulated ones. A zero or close-to-zero P-value for any

exon indicates that the number of simulations is insuffi-

cient for generating a reliable P-value estimate. For all

exons with a P-value of smaller than three times the pre-

cision, the number of simulations is increased by 10-fold in

a new round of simulation, which increases the precision

of the P-value estimate from 0.01 to 0.001. This adaptive

sampling procedure is repeated for multiple rounds.

The default setting of MATS is to repeat this procedure

for at most six rounds to reach the highest P-value preci-

sion of 10?7, but this parameter can be adjusted by users.

This adaptive sampling procedure enables us to selectively

increase the precision and running time for exons with

significant (i.e. small) P-values, thus reducing the overall

running time needed for all exons.

After we obtain the P-values of all exons, we apply

the classic Benjamini–Hochberg method (24) on these

P-values to obtain the FDR values.

{Psim

ij

}as

1

M

PM

j¼1

i

? Psim

ijÞ.

Detecting switch-like differential alternative splicing

MATS offers the flexibility for testing different types of

hypotheses on the differential alternative splicing pattern.

An analysis of considerable biological interest is to identify

exons with the extreme ‘switch-like’ differential alter-

native splicing pattern, i.e. ð 1< s and 2> 1 ? sÞ or

ð 1> 1 ? s and 2< sÞ where s is a user-defined param-

eter between 0 and 1/2. For example, if we set s as 1/3, we

test if the exon inclusion level of an exon switches from

less than 1/3 in one sample to more than 2/3 in the other

sample. Such an extreme switch of exon inclusion levels

between conditions is a strong indicator of functional alter-

nativesplicingevents(3,22).Inthe‘switch-like’test,MATS

considers the null hypothesis that the 1and 2are outside

oftheregiondefinedby

or ð 1> 1 ? s and 2< sÞ. The values of 1 and 2

under the null hypothesis (H0) and the alternative hypoth-

esis (H1) for the ‘switch-like’ test are illustrated graphically

in Figure 2B. The same MCMC and simulation procedures

for testing j 1? 2j > c can be used to calculate the

Bayesian posterior probability, P-value, and FDR for

‘switch-like’ differential alternative splicing.

ð 1< s and 2> 1 ? sÞ

Exon–exon junction database of human genes

We constructed a database of exon–exon junctions in

human genes using the Ensembl transcript annotations

(release 57) (28). The database includes all known exon–

exon junctions observed in Ensembl transcripts, as well as

PAGE 5 OF 13Nucleic AcidsResearch, 2012, Vol.40,No. 8e61

Page 6

hypothetical exon-exon junctions obtained by all possible

pairwise fusions of exons within genes. In total, the data-

base contains ?3.5 million exon–exon junctions. Each

exon–exon junction sequence is 84bp long with 42bp

from the 30-end of the upstream exon and 42bp from

the 50-end of the downstream exon. This exon–exon junc-

tion database is available for download from the MATS

website http://intron.healthcare.uiowa.edu/MATS/.

RNA-Seq analysis of ESRP1 regulated differential

alternative splicing events in the MDA-MB-231 breast

cancer cell line

MDA-MB-231 cells were maintained and retrovirally

transduced by a cDNA encoding the epithelial-specific

splicing factor ESRP1 or the empty vector (EV) control

as described previously (23,29). Sequencing libraries were

preparedusingthemRNA-Seq

(Illumina) according to the manufacturer’s instructions.

Ten micrograms of total RNA was used to prepare

polyA RNA for fragmentation followed by cDNA synthe-

sis with random hexamers and ligation to Illumina

adaptor sequences. The samples underwent an RNA

quality assurance check and were quantified using an

Agilent 2100 Bioanalyzer, loaded onto flow-cells for cluster

generation, and sequenced on an Illumina Genome

Analyzer II using single-end protocol to generate 76bp

reads (Illumina). The resulting RNA-Seq dataset consisted

of 256 million single-end reads, including 136 million

reads for the ESRP1 sample and 120 million reads for

the EV sample.

During the quality assessment of our 76bp single-end

RNA-Seq data, we found that the first two 25bp segments

of these reads had a high mapping rate to the human

genome, while the 3rd 25bp segment had a much lower

mapping rate (data not shown). This is likely due to the

increased sequencing error rate near the 30-end of

the Illumina RNA-Seq reads. Thus, we decided to use

the first 50bp of each read for mapping and subsequent

analysis. We mapped RNA-Seq reads to the human

genome (hg19) and the exon–exon junction database,

using the software Bowtie (30) allowing up to 3bp

mismatches. Each mapped exon–exon junction read

required at least 8bp from each side of the exon–exon

junction. We further removed exon–exon junction reads

that mapped to either the human genome (hg19) or

multiple exon–exon junctions. For all identified alterna-

tively spliced cassette exons, the exon–exon junction

counts were used as the input for MATS.

SamplePrepKits

Illumina Human Body Map 2.0 data on 16 human tissues

We obtained a paired-end RNA-Seq data set from

Illumina, with ?80 million read pairs per tissue for 16

human tissues (adipose, adrenal, brain, breast, colon,

heart, kidney, liver, lung, lymph node, ovary, prostate,

skeletal muscle, testes, thyroid and white blood cells).

This data set was referred to as the ‘Human Body Map

2.0’ by Illumina. For each paired-end read (50bp?2), we

mapped each end to the human genome (hg19) and the

exon–exon junction database, using the software Bowtie

(30) allowing up to 3bp mismatches. The final mapping

location of the paired-end read was determined by

requiring that the two ends be mapped in the opposite

orientation tothesame

maximum genomic distance of 50kb between the two

ends (to allow introns between the two mapped ends).

genomicregion,witha

RT–PCR validation

Quantitation of alternative splicing was performed using

standard RT–PCR incorporating radiolabeled dCTP or

fluorescently labeled primers, or high-throughput RT–

PCR at the Universite ´ de Sherbrooke as described

(23,31). Since we used MATS to test j 1? 2j > 0:1, we

defined a differential alternative splicing event as validated

if the RT–PCR-based exon inclusion levels differed by at

least 10% between the two samples with the direction of

change matching the RNA-Seq prediction.

RESULTS

Multivariate uniform prior in MATS

MATS uses a multivariate uniform distribution to model

the joint prior of exon inclusion levels of alternatively

spliced cassette exons in two samples. This is different

from and more general than the independent uniform

priors used by previous methods (11,16). Note that the

multivariate uniform prior includes the independent

uniform prior model as a special case (?=0). These two

types of prior distributions have distinct underlying as-

sumptions on the alternative splicing patterns of different

biological conditions. Intuitively, the multivariate uniform

prior allows the modeling of similarity in alternative

splicing patterns between two samples (using the correl-

ation parameter ?). In contrast, the independent uniform

priors assume that the global splicing pattern of one

sample is independent of the other sample. To determine

whether the multivariate uniform prior is appropriate and

able to capture the correlation pattern in the data, we

analyzed two RNA-Seq data sets covering diverse tissues

and cell types.

We first compared the alternative splicing profiles of a

single cell line subject to two different treatments. The

data set came from our deep single-end RNA sequencing

of a human breast cancer cell line (MDA-MB-231) with

ectopic expression of the epithelial-specific splicing factor

ESRP1 or an empty vector (EV) control (see ‘Materials

and Methods’ section). ESRP1 encodes a master cell-type

specific regulator of alternative splicing that controls a

global epithelial-specific splicing network (23,29). In the

MDA-MB-231 cell line, the ectopic expression of ESRP1

drives coordinated switches of ESRP1-regulated exons

towardsanepithelialsplicing

provides an excellent system for testing algorithms of

alternative splicing analysis. We generated 136 million

single-end reads on the ESRP1 sample and 120 million

single-end reads on the EV sample. We identified a total

of 18859 alternatively spliced cassette exons in this data

set. Pan and colleagues previously demonstrated that

RNA-Seq can reliably estimate the exon inclusion levels

of alternatively spliced exons, when the sequencing

coverage reaches at least 20 reads for one of the three

signature(23).This

e61 Nucleic Acids Research, 2012,Vol. 40,No. 8PAGE 6 OF 13

Page 7

exon-exon junctions (4). Therefore, to assess the global

correlation in alternative splicing patterns between these

two samples (ESRP1 and EV), we restricted our analysis

to 12890 alternatively spliced cassette exons with at least

20 reads mapped to one of the three exon-exon junctions

in both samples. We observed a high correlation in

the exon inclusion levels of these exons between the

ESRP1 and EV samples (Pearson correlation r=0.95,

P<2.2e-16; Figure 3A). In contrast, the exon inclusion

levels simulated from two independent uniform priors

had no correlation between the two samples (Pearson

correlation r=0; Figure 3B), contradicting with the

real data.

To assess if the multivariate uniform prior is capable of

modeling the correlation pattern in the data, we used our

MCMC procedure to obtain the estimate of the correl-

ation parameter ? on the real ESRP data set. As the

control, we analyzed 10000 simulated data sets using

exon inclusion levels simulated from two independent

uniform priors, in which no correlation existed between

the two samples. For the ESRP data, our MCMC proced-

ure obtained an estimate of ? of 0.93, consistent with the

high overall positive correlation observed in the data. In

contrast, the estimates of ? on the 10000 simulated data

sets were close to 0, indicating no correlation between the

two samples (Figure 3C). These results suggest that the

multivariate uniform prior model is flexible enough for

both situations, and that the MCMC procedure is

capable of obtaining an estimate of the parameter ? that

reflects the degree of correlation in the data.

To assess if the pattern observed in the MDA-MB-231

cell line holds true when we compare more distantly

related samples of different tissue origins, we analyzed

the Illumina Human Body Map 2.0 data on 16 human

tissues (see ‘Materials and Methods’ section). We per-

formed pairwise comparisons of alternative splicing

profiles of all possible tissue pairs. Between any two

tissues, we observed a high overall correlation in the

estimated exon inclusion levels of alternatively spliced

cassette exons, with the Pearson correlation coefficient

ranging from 0.89 to 0.97 (Supplementary Figure S1).

These data further justify the use of the multivariate

uniform prior, even for the comparison between more

divergent samples representing different tissue types.

Simulation study of MATS

We evaluated the performance of MATS with a simula-

tion study.Specifically, we

RNA-Seq data set with a mixture of data points repre-

senting non-differentially spliced exons and differentially

spliced exons. The threshold of the exon inclusion level

difference between two samples was set as 10% in our

simulation study (i.e. j 1? 2j > 0:1). We generated

data for non-differentially spliced exons by sampling the

exon inclusion level of an exon in sample 1 from a uniform

(0, 1) distribution, and randomly added or subtracted a

small variation drawn from a uniform (0, 0.1) distribution

to obtain the exon inclusion level in sample 2. We

generated datafordifferentially

sampling the exon inclusion level of an exon in sample 1

from a uniform (0, 1) distribution, and randomly added or

subtracted a large variation drawn from a uniform (0.1, 1)

distribution to obtain the exon inclusion level in sample 2.

For all simulated exons, if the variation added to or sub-

tracted from the exon inclusion level in sample 1 caused

the exon inclusion level in sample 2 to be above 1 or below

0, the sampling step for the variation was repeated until

the exon inclusion level in sample 2 was within the [0,1]

range. In the simulated data, we generated a total of 5000

data points in which 95% represented non-differentially

spliced exons and 5% represented differentially spliced

exons (Figure 4A). After the exon inclusion levels were

simulated for 5000 exons, we generated 5 simulated data

sets, by setting the total inclusion+skipping isoform

junction counts per exon and sample as 100, 200, 500,

generatedasimulated

splicedexonsby

Figure 3. The multivariate uniform prior can model the between-sample correlation pattern in the RNA-Seq data. (A) The scatter plot of the

estimated exon inclusion levels of 12890 alternatively spliced cassette exons in the ESRP1 and EV samples. Only exons with at least 20 reads mapped

to one of the three exon–exon junctions in both samples are included in the plot. (B) The scatter plot of the exon inclusion levels in two samples

simulated from two independent uniform priors. In (A and B), the two red lines define the area where j 1? 2j ? 0:1. (C) The MCMC estimate of

the correlation parameter ? can capture the correlation pattern in the data. For the ESRP data, ^ ? is 0.93 (the red vertical line). For the 10000

simulated data sets from independent uniform priors, ^ ? is distributed close to zero.

PAGE 7 OF 13Nucleic AcidsResearch, 2012, Vol.40,No. 8 e61

Page 8

1000 and 2000 respectively. The inclusion isoform count

of an exon in a sample was then calculated as the product

of its simulated exon inclusion level and the total junction

count. After the simulation data sets were generated, we

used MATS to calculate the P-value and FDR of each

exon, and compared the estimated FDRs to the true

FDRs. As shown in Figure 4B–F, although the estimated

FDRs were generally more conservative (i.e. with higher

values) than the true FDRs, the overall curve of the

estimated FDR followed the trend of the true FDR

curve, especially for exons ranked by MATS among the

top 250 (i.e. the number of true positives in our simulated

Figure 4. Simulation study of MATS. (A) Simulated exon inclusion levels of 5000 exons in two samples. A total of 95% of the data points are

simulated from the null hypothesis (j 1? 2j ? 0:1) and 5% are simulated from the alternative hypothesis (j 1? 2j > 0:1). (B–F) MATS FDR

estimates on simulated data with the exon inclusion levels from (A) and the total junction count per exon and sample as 100 (B), 200 (C), 500 (D),

1000 (E) and 2000 (F). In each panel, exons are rank sorted by MATS FDR estimates in ascending order. The zoomed-in figure shows the FDR

estimates of the top 250 exons by MATS.

e61 Nucleic Acids Research, 2012,Vol. 40,No. 8PAGE 8 OF 13

Page 9

data set). We observed a sharp increase in the estimated

FDR when the MATS rank of differential alternative

splicing approached 250, consistent with the total

number oftruepositives

Moreover, the number of exons with MATS FDR value

of close to 0 increased progressively with increasing

simulated junction counts (see the zoomed-in figures of

Figure 4B–F), reflecting the influence of RNA-Seq depth

on the sensitivity of detecting differential alternative

splicing events. We note that non-differentially spliced

exons constitute 95% of the simulated data points. For

such exons, the correct FDR estimates should be high

FDR values.

To further evaluate the performance of the MATS algo-

rithm especially the benefit of the correlation parameter ?,

we conducted another simulation study to compare

MATS with a simplified MATS Bayesian model in

which ? is fixed at 0 (i.e. independent prior), as well as

the Fisher exact test. As in the previous simulation study,

we generated 5000 data points in which 95% represented

non-differentially spliced exons and 5% represented dif-

ferentially spliced exons. To mimic the overall distribution

of junction counts in real data sets, for each simulated

exon its total junction counts (i.e. inclusion+skipping

isoform junction counts) in sample 1 and 2 respectively

were randomly sampled from the MDA-MB-231 ESRP1

data set by taking the counts of a randomly selected alter-

natively spliced cassette exon in the ESRP1 and EV

samples. For each of the three methods tested, we

calculated the true positive rate and false positive rate

under sliding P-value cutoffs from 0 to 1. We then

generated the receiver operating characteristic (ROC)

curve for each method as the true positive rate versus

false positive rate plot, and calculated the area under

curve (AUC) for each method (Figure 5). MATS had

the highest AUC of 0.96, significantly better than the

inthesimulateddata.

simplified MATS Bayesian model with ? ¼ 0 (AUC=

0.88, DeLong’s Test P=6.8e-8). MATS also significantly

outperformed the Fisher

DeLong’s Test P<2.2e-16). Additionally, we note that

even the simplified MATS Bayesian model (with ? ¼ 0)

outperformed the Fisher exact test in the most critical

area of the ROC curve where the false positive rate was

low (Figure 5). This indicates that by testing for difference

(with a threshold) instead of equality, the test statistics is

better at separating true positives from false positives.

exacttest (AUC=0.90,

MATS analysis of ESRP1-regulated differential

alternative splicing

To evaluate the performance of MATS on a real data set,

we used MATS to detect ESRP1-regulated differential al-

ternative splicing events using our RNA-Seq data on the

MDA-MB-231 cell line with ectopic expression of ESRP1

or an empty vector (EV) control. For each of the 18859

alternatively spliced cassette exons (defined as exons with

at least one inclusion read and one skipping read in these

two samples), we calculated the Bayesian posterior prob-

ability, P-value and FDR for j 1? 2j > 0:1. Among 240

exons with MATS FDR of <10%, all (100%) had poster-

ior probability of >0.85, including 239 (99.6%) and 234

(97.5%) with posterior probability of >0.9 and >0.95 re-

spectively. Figure 6 illustrates a differentially spliced exon

(exon 7 of SPNS1) identified by MATS. Based on the

RNA-Seq read counts we estimated an exon inclusion

level of 77% in the EV sample and 27% in the ESRP1

sample, with a FDR value of 4.6e-4 (Figure 6A). These

predictions matched RT-PCR results of SPNS1 exon 7

splicing in these two samples (Figure 6B).

To assess the overall accuracy of our FDR estimates, we

selected 164 exons covering a broad range of MATS FDR

values (Supplementary Table S1) and tested their splicing

patterns by RT–PCR. Of all the exons tested by RT–PCR,

111 exons had at least 10% difference in the exon inclu-

sion levels between the two samples with the direction of

change matching the RNA-Seq predictions. This yielded

an overall validation rate of 68%. To assess whether the

validation rate correlated with MATS FDR estimates,

we divided the full list of 164 exons into four cohorts ac-

cording to the estimated FDR values, and calculated the

RT–PCR validation rate for each cohort. We observed a

progressive decrease in the RT–PCR validation rate for

cohorts with increasing FDR values (Figure 7). The first

cohort had 92 exons with FDR estimates between 0 to

10%. In this cohort, 79 exons were validated by

RT–PCR as differentially spliced, yielding a high valid-

ation rate of 86%. The second, third and fourth cohorts

corresponded to exons with FDR estimates between 10%

and 30%, between 30% and 60%, and between 60% and

100%. These three cohorts had RT–PCR validation rates

of 73%, 55% and 36%, respectively (Figure 7). These

results indicate that MATS can generate experimentally

meaningful FDR estimates to help biologists with the in-

terpretation of RNA-Seq predictions and the design of

follow-up experiments. There was a sharp increase in the

estimated FDR value after the initial list of top 240–406

exons (Figure 7), with ?98% of the exons having a FDR

Figure 5. Simulation study to compare MATS, a simplified MATS

Bayesian model in which ? is fixed at 0 (i.e. independent prior), and

the Fisher exact test. MATS significantly outperforms the other two

methods based on the AUC of the ROC curve (i.e. the true positive

rate versus false positive rate plot).

PAGE 9 OF 13Nucleic AcidsResearch, 2012, Vol.40,No. 8 e61

Page 10

of ?90%. This was similar to the shape of the FDR dis-

tribution in the simulation study (Figure 4), probably re-

flecting the number of ESRP1-regulated exons in the

human genome as well as the percentage of which that

can be detected at the current RNA-Seq depth. Of note,

among the 164 exons tested by RT–PCR, 17 had a MATS

FDR of 100%. Only 1 of the 17 exons had more than 10%

change in the RT–PCR estimated exon inclusion levels

with the direction of change matching the RNA-Seq pre-

diction, yielding a low validation rate of only 6% that

closely matched the estimated FDR. This indicates that

MATS can provide reliable FDR estimates for the full

range of possible FDR values.

Since our exon–exon junction database includes all

known exon-exon junctions observed in Ensembl tran-

scripts, as well as hypothetical exon–exon junctions

obtained by all possible pairwise fusions of exons within

genes, we can detect and analyze novel exon skipping

events not supported by existing transcript annotations.

This is important considering the prevalence of tissue-

and condition-specific alternative splicing (32). Of all

18859 alternatively spliced cassette exons, 5373 represent

known events and 13486 represent novel events. Of the

240 significant events with MATS FDR<10%, 140 repre-

sent known events while 100 represent novel events. The

percentage of events called as differentially spliced is sig-

nificantly higher for known events (2.6%; 140/5373) than

for novel events (0.7%; 100/13486). Interestingly, of the 92

RT–PCR tested cassette exons with FDR<10%, the RT–

PCR validation rate of differential alternative splicing is

higher for novel events (98%; 43/44) than for known

events (75%; 36/48).

The MATS algorithm can be naturally extended to

other types of alternative splicing events such as alterna-

tive 50or 30splice sites and mutually exclusive exon usage.

In the analysis of alternative 50or 30splice sites, the counts

of junction reads that uniquely support the two competing

splice sites can serve as the input for MATS. These counts

can be used to estimate the ‘inclusion level’ of any specific

splice site. All subsequent analysis steps are identical to

the analysis of differential exon skipping. To illustrate

this, we applied MATS to 1571 alternative 50splice site

events and 2383 alternative 30splice site events in the

ESRP1 data set. With a FDR cutoff of 5%, 13 events (9

alternative 50splice sites and 4 alternative 30splice sites)

were identified to undergo significant differential splicing

(>10% change) between the ESRP1 and EV samples. The

small number of detected differential alternative 50or 30

splice sites is consistent with an early study using the

Affymetrix exon 1.0 array (33). One possible explanation

is that ESRP1 regulates a small number of such events in

the transcriptome. Nonetheless, we tested five events by

RT–PCR, of which three were validated to have at least

10% change in splice site inclusion level (Supplementary

Table S2). Supplementary Figure S2 shows the example of

a validated differential alternative 5’ splice site event in

exon 4 of HNRNPH3.

MATS analysis of switch-like alternative splicing between

brain and 15 other tissues

MATS has the flexibility to detect exons with the extreme

‘switch-like’differentialalternativesplicingpattern

Figure 6. RNA-Seq and RT–PCR analysis of SPNS1 exon 7 splicing. (A) RNA-Seq junction counts and MATS result of SPNS1 exon 7 in the EV

and ESRP1 samples. (B) RT–PCR result of SPNS1 exon 7 in the EV and ESRP1 samples.

Figure 7. RT–PCR validation of 164 exons covering a broad range of

MATS FDR values. All exons analyzed by MATS are rank sorted by

FDR estimates (y-axis) in ascending order. The 164 exons tested by

RT–PCR are divided into four non-overlapping cohorts according to

the FDR estimates. The validation rate for each cohort is shown.

e61Nucleic Acids Research, 2012,Vol. 40,No. 8PAGE 10 OF 13

Page 11

(see ‘Materials and Methods’ section). To illustrate this

function, we used MATS to detect switch-like differential

alternative splicing events between the brain and each of

the 15 other tissues in the Illumina Human Body Map 2.0

data set. For each pairwise comparison, we tested if the

exon inclusion level of an exon switches from less than 1/3

in one tissue to more than 2/3 in the other tissue (i.e.

ð 1< 1=3 and 2> 2=3Þ or ð 1> 2=3 and 2< 1=3Þ.

With a FDR cutoff of <50%, a total of 229 exons were

identified to have the switch-like differential alternative

splicing pattern between the brain and at least one other

tissue. Prior studies have revealed sequence features of

such ‘tissue-switched’ cassette exons characteristic of func-

tional alternative splicing events (3,22). A unique feature

of tissue-switched exons is that they are much more likely

to be exact multiples of 3nt in length, thus alternative

splicing adds or removes a modular peptide segment of

the final protein product while preserving the downstream

open reading frame (i.e. ‘frame-preserving’, as opposed

to ‘frame-switching’ for exons not exact multiples of 3nt

in length). Consistent with these findings, of the 229

switch-like exons detected by MATS between brain and

15 other tissues, 70% are frame-preserving, compared to

42% for other alternatively spliced cassette exons without

switch-like differential alternative splicing (P=0; see

Supplementary Figure S3).

DISCUSSION

We present MATS, a new method to detect differential

alternative splicing events from RNA-Seq data. A major

advantage of MATS over existing methods is that it allows

flexible hypothesis testing of differential alternative

splicing patterns. Most of the published work attempted

to identify differential alternative splicing events by testing

the equality of the exon inclusion levels between samples

(11,15,16,18–20). Some also applied a secondary filter

(without statistical testing) on the change in the estimated

exon inclusion levels (20). MATS provides a Bayesian

statistical framework to directly test the hypothesis and

evaluate the statistical significance that the absolute dif-

ference in exon inclusion levels between two samples

exceeds any user-defined threshold. This allows research-

ers to select the magnitude of splicing changes suitable

for specific research goals in a rigorous statistical setting.

We assessed the performance of MATS using simulated

data and real RNA-Seq data sets. In the RNA-Seq

analysis of ESRP1 regulated alternative splicing, we

obtained a high RT–PCR validation rate of 86% for can-

didate exons with MATS FDR <10%. Additionally, over

the full list of RT–PCR tested exons, the MATS FDR

estimates matched well with the experimental validation

rate (Figure 7). The MATS framework is also applicable

to other null hypotheses of interest. For example,

MATS can be used to test the hypothesis that an exon

exhibits the extreme ‘switch-like’ differential alternative

splicing pattern.

A novel feature of MATS is the multivariate uniform

prior that models the between-sample correlation in exon

splicing patterns. In both the ESRP data and the Human

Body Map 2.0 data, the degree of correlation between

samples is high, resulting in a high estimated value of

the correlation parameter ? for the multivariate prior

model. The high between-sample correlation observed in

real RNA-Seq data is consistent with the mechanism of

splicing regulation in eukaryotic cells. Splicing is a

complex process mediated by extensive interactions

among cis regulatory elements and trans acting regulators

(34). Most splicing regulators may control the splicing of

up to several hundred exons via sequence-specific protein–

RNA interactions (35). Perturbing a specific component of

the splicing regulatory pathway usually changes the

splicing activity of a small subset of alternatively spliced

exons, while the majority of alternatively spliced exons

remain unaffected. We also note that when no correlation

exists in the data (see Figure 3 for the simulated data), the

estimate of ? by the MCMC procedure is close to zero.

Taken together, our analysis indicates that the multivari-

ate uniform prior model is flexible enough to accommo-

date different degrees of between-sample correlation

in the RNA-Seq data. Moreover, our simulation study

(Figure 5) indicates that incorporating the correlation par-

ameter ? in the MATS model improves the performance

of the algorithm.

The MATS software and documentation as well as

the raw ESRP1 RNA-Seq data are freely available

at http://intron.healthcare.uiowa.edu/MATS/. The scripts

provided online include the core MATS program to cal-

culate the posterior probability, P-value and FDR of dif-

ferential alternative splicing from the input junction

counts, as well as accessory scripts to generate junction

counts and detect alternative splicing events from raw

RNA-Seq data. To facilitate data analysis, we also

provide databases of pre-compiled exon-exon junctions

based on the Ensembl (28) or UCSC Known Genes (36)

annotations. We suggest that MATS can be used either as

a stand-alone software for differential alternative splicing

analysis of RNA-Seq data, or as part of an existing

RNA-Seq analysis pipeline to calculate the statistical sig-

nificance of a user-defined differential splicing pattern

using junction counts generated by other mapping proced-

ures or transcript annotation databases. We note that

a number of recent studies have reported biases in

RNA-Seq data such as the non-uniform distribution of

RNA-Seq reads along mRNA transcripts, and have

proposed methods to adjust raw RNA-Seq read counts

by correcting for such biases (18,37–41). Additionally, it

has been demonstrated that in paired-end RNA-Seq, the

distribution of insert size between the two ends can be

utilized to improve the assignment of reads to specific

transcript isoforms (16). Thus, it is possible to use an ap-

propriate method to adjust raw RNA-Seq read counts and

refine the counts of isoform-specific junctions, prior to the

hypothesis testing of differential alternative splicing by

MATS. It should also be noted that although the

analysis in this manuscript is mostly focused on exon

skipping events (i.e. differential inclusion/skipping of an

entire exon), exon skipping is only one subtype of alter-

native splicing events. The MATS algorithm can be

readily applied to junction counts generated for other

types of alternative splicing events, as illustrated for

PAGE 11 OF 13Nucleic AcidsResearch, 2012, Vol.40,No. 8e61

Page 12

alternative 50or 30splice sites on the ESRP1 data set

(Supplementary Figure S2).

MATS currently performs two-group comparison of

two samples, with one sample per group without

within-group replicates. This is the typical experimental

setup in most published RNA-Seq studies of alternative

splicing including studies of splicing regulators (16,20,42),

largely due to the high cost of RNA-Seq to achieve suffi-

cient depth for splicing analysis. However, as the cost of

high-throughput sequencing continues to decline, we an-

ticipate that it will soon become feasible and common for

researchers toincorporate

RNA-Seq studies of alternative splicing (43). Medical re-

searchers may soon be able to generate RNA-Seq data

across a large number of healthy and diseased specimens,

with the depth sufficient for quantifying splicing in each

individual sample. Thus, an important future direction is

to extend the statistical framework to incorporate the use

of RNA-Seq replicates in detecting differential alternative

splicing.

biological replicatesin

SUPPLEMENTARY DATA

Supplementary Data are available at NAR Online:

Supplementary Figures S1–S3 and Supplementary Tables

S1–S2.

ACKNOWLEDGEMENTS

We wish to thank Keyan Zhao for comments, and Collin

Tokheim for technical assistance. We thank Gary Schroth

for early access to the Illumina Human Body Map 2.0

data. We thank David Eichmann, Ben Rogers and the

Universityof IowaInstitute

Translational Science (NIH grant UL1RR024979) for

computer support.

forClinicaland

FUNDING

National

P30DK054759

R01CA120988 to J.H.); National Science Foundation

(DMS-0805491, Career award DMS-1055286 to Q.Z.); a

junior faculty grant from the Edward Mallinckrodt Jr

Foundation (to Y.X.); National Institutes of Health T32

postdoctoral fellow training grant (T32HL007638 to

J.W.P.). Fundingfor open

R01GM088342.

Institutes of Health

R01GM088809

(R01GM088342 and

toY.X., toR.P.C.,

access charge:NIH

Conflict of interest statement. None declared.

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