#### Authors

Ofer Shai, Brendan J. Frey, Quaid Morris, Qun Pan, Christine Misquitta, Benjamin Blencowe

#### Abstract

Alternative splicing (AS) is an important and frequent step in mammalian gene expression that allows a single gene to specify multiple products, and is crucial for the regulation of fundamental biological processes. The extent of AS regulation, and the mechanisms involved, are not well un- derstood. We have developed a custom DNA microarray platform for surveying AS levels on a large scale. We present here a generative model for the AS Array Platform (GenASAP) and demonstrate its utility for quantifying AS levels in different mouse tissues. Learning is performed using a variational expectation maximization algorithm, and the parame- ters are shown to correctly capture expected AS trends. A comparison of the results obtained with a well-established but low through-put experi- mental method demonstrate that AS levels obtained from GenASAP are highly predictive of AS levels in mammalian tissues.

1 Biological diversity through alternative splicing

Current estimates place the number of genes in the human genome at approximately 30,000, which is a surprisingly small number when one considers that the genome of yeast, a single- celled organism, has 6,000 genes. The number of genes alone cannot account for the com- plexity and cell specialization exhibited by higher eukaryotes (i.e. mammals, plants, etc.). Some of that added complexity can be achieved through the use of alternative splicing, whereby a single gene can be used to code for a multitude of products.

Genes are segments of the double stranded DNA that contain the information required by the cell for protein synthesis. That information is coded using an alphabet of 4 (A, C, G, and T), corresponding to the four nucleotides that make up the DNA. In what is known as the central dogma of molecular biology, DNA is transcribed to RNA, which in turn is translated into proteins. Messenger RNA (mRNA) is synthesized in the nucleus of the cell and carries the genomic information to the ribosome. In eukaryotes, genes are generally comprised of both exons, which contain the information needed by the cell to synthesize proteins, and introns, sometimes referred to as spacer DNA, which are spliced out of the pre-mRNA to create mature mRNA. An estimated 35%-75% of human genes [1] can be

                                                                                                                            C                    A                C                                                                                                                                 1                                           2                     (a)                   C                              A                        C                                                 1                                                      2                                                                                                                                            C                   C                                                                                                                                            1                    2

C    A          C                   C A          C                                                                                                                            1         3'         2              1      5'         2                     (b)    C         A                         C                                                 C                                 1              3'                   2         C1                  A5'                 2                                                                                                                                            C                   C                                                                                                                                            1                    2

C                    A                C                                                                                                                                 1                         1                 2                     (c)              C                         A              A                             C                                           1                         1              2                             2                                                                                                                                 C                    A                C                                                                                                                                 1                         2                 2

C                   C                                                                                                                                            1                    2                     (d)                              C                                  C                                                           1                                  2                                                                                                                                 C                                     C                                                                                                                                 1                                           2


Figure 1: Four types of AS. Boxes represent exons and lines represent introns, with the possible splicing alternatives indicated by the connectors. (a) Single cassette exon inclusion/exclusion. C1 and C2 are constitutive exons (exons that are included in all isoforms) and flank a single alternative exon (A). The alternative exon is included in one isoform and excluded in the other. (b) Alternative 3' (or donor) and alternative 5' (acceptor) splicing sites. Both exons are constitutive, but may con- tain alternative donor and/or acceptor splicing sites. (c) Mutually exclusive exons. One of the two alternative exons (A1 and A2) may be included in the isoform, but not both. (d) Intron inclusion. An intron may be included in the mature mRNA strand.

spliced to yield different combinations of exons (called isoforms), a phenomenon referred to as alternative splicing (AS). There are four major types of AS as shown in Figure 1. Many multi-exon genes may undergo more than one alternative splicing event, resulting in many possible isoforms from a single gene. [2]

In addition to adding to the genetic repertoire of an organism by enabling a single gene to code for more than one protein, AS has been shown to be critical for gene regulation, con- tributing to tissue specificity, and facilitating evolutionary processes. Despite the evident importance of AS, its regulation and impact on specific genes remains poorly understood. The work presented here is concerned with the inference of single cassette exon AS levels (Figure 1a) based on data obtained from RNA expression arrays, also known as microar- rays.

1.1 An exon microarray data set that probes alternative splicing events

Although it is possible to directly analyze the proteins synthesized by a cell, it is easier, and often more informative, to instead measure the abundance of mRNA present. Traditionally, gene expression (abundance of mRNA) has been studied using low throughput techniques (such as RT-PCR or Northern blots), limited to studying a few sequences at a time and making large scale analysis nearly impossible.

In the early 1990s, microarray technology emerged as a method capable of measuring the expression of thousands of DNA sequences simultaneously. Sequences of interest are de- posited on a substrate the size of a small microscope slide, to form probes. The mRNA is extracted from the cell and reverse-transcribed back into DNA, which is labelled with red and green fluorescent dye molecules (cy3 and cy5 respectively). When the sample of tagged DNA is washed over the slide, complementary strands of DNA from the sample hy- bridize to the probes on the array forming A-T and C-G pairings. The slide is then scanned and the fluorescent intensity is measured at each probe. It is generally assumed that the intensity measure at the probe is linearly related to the abundance of mRNA in the cell over a wide dynamic range.

Despite significant improvements in microarray technologies in recent years, microarray data still presents some difficulties in analysis. Low measurements tend to have extremely low signal to noise ratio (SNR) [7] and probes often bind to sequences that are very similar, but not identical, to the one for which they were designed (a process referred to as cross-

                   C                        A                    C                         1                                            2

C                       A                    C         3 Body probes                          1                                                2

C :A           A:C                                        1                   2

C              A               C              2 Inclusion junction probes                                  1                              2

C :C                                                 1    2

C              C                        1 Exclusion junction probe                                          1                2


Figure 2: Each alternative splicing event is studied using six probes. Probes were chosen to measure the expression levels of each of the three exons involved in the event. Additionally, 3 probes are used that target the junctions that are formed by each of the two isoforms. The inclusion isoform would express the junctions formed by C1 and A, and A and C2, while the exclusion isoform would express the junction formed by C1 and C2

hybridization). Additionally, probes exhibit somewhat varying hybridization efficiency, and sequences exhibit varying labelling efficiency.

To design our data sets, we mined public sequence databases and identified exons that were strong candidates for exhibiting AS (the details of that analysis are provided elsewhere [4, 3]). Of the candidates, 3,126 potential AS events in 2,647 unique mouse genes were selected for the design of Agilent Custom Oligonucleotide microarray. The arrays were hybridized with unamplified mRNA samples extracted from 10 wild-type mouse tissues (brain, heart, intestine, kidney, liver, lung, salivary gland, skeletal muscle, spleen, and testis). Each AS event has six target probes on the arrays, chosen from regions of the C1 exon, C2 exon, A exon, C1:A splice junction, A:C2 splice junction, and C1:C2 splice junction, as shown in Figure 2.

2 Unsupervised discovery of alternative splicing

With the exception of the probe measuring the alternative exon, A (Figure 2), all probes measure sequences that occur in both isoforms. For example, while the sequence of the probe measuring the junction A:C1 is designed to measure the inclusion isoform, half of it corresponds to a sequence that is found in the exclusion isoform. We can therefore safely assume that the measured intensity at each probe is a result of a certain amount of both isoforms binding to the probe. Due to the generally assumed linear relationship between the abundance of mRNA hybridized at a probe and the fluorescent intensity measured, we model the measured intensity as a weighted sum of the overall abundance of the two isoforms.

A stronger assumption is that of a single, consistent hybridization profile for both isoforms across all probes and all slides. Ideally, one would prefer to estimate an individual hy- bridization profile for each AS event studied across all slides. However, in our current setup, the number of tissues is small (10), resulting in two difficulties. First, the number of parameters is very large when compared to the number of data point using this model, and second, a portion of the events do not exhibit tissue specific alternative splicing within our small set of tissues. While the first hurdle could be accounted for using Baysian parameter estimation, the second cannot.

2.1 GenASAP - a generative model for alternative splicing array platform

Using the setup described above, the expression vector x, containing the six microarray measurements as real numbers, can be decomposed as a linear combination of the abun- dance of the two splice isoforms, represented by the real vector s, with some added noise: x = s + noise, where is a 6 2 weight matrix containing the hybridization profiles for

                                                       s                                     s                                                            1                                          2

x^                  x                                                 ^               x^              x^                         x                                                                                                            ^               x^                                   C                  C                A          C :A                           A:C              C :C                                    1                  2                               1                               2           1    2

r

x                   x               x               x                          x               x                                  C                   C               A           C :A                           A:C              C :C                                    1                  2                               1                               2           1    2

o                   o               o               o                          o               o                               C                      C           A               C :A                           A:C         C :C                                    1                  2                               1                               2           1     2

n    2


Figure 3: Graphical model for alternative splicing. Each measurement in the observed expression profile, x, is generated by either using a scale factor, r, on a linear combination of the isoforms, s, or drawing randomly from an outlier model. For a detailed description of the model, see text.

the two isoforms across the six probes. Note that we may not have a negative amount of a given isoform, nor can the presence of an isoform deduct from the measured expression, and so both s and are constrained to be positive.

Expression levels measured by microarrays have previously been modelled as having expression-dependent noise [7]. To address this, we rewrite the above formulation as

                                                       x = r(s + ),                                                                    (1)


where r is a scale factor and is a zero-mean normally distributed random variable with a diagonal covariance matrix, , denoted as p() = N (; 0, ). The prior distribution for the abundance of the splice isoforms is given by a truncated normal distribution, denoted as p(s) N (s, 0, I)[s 0], where [] is an indicator function such that [s 0] = 1 if i, si 0, and [s 0] = 0 otherwise.

Lastly, there is a need to account for aberrant observations (e.g. due to faulty probes, flakes of dust, etc.) with an outlier model. The complete GenASAP model (shown in Figure 3) accounts for the observations as the outcome of either applying equation (1) or an outlier model. To avoid degenerate cases and ensure meaningful and interpretable results, the number of faulty probes considered for each AS event may not exceed two, as indicated by the filled-in square constraint node in Figure 3.

The distribution of x conditional on the latent variables, s, r, and o, is:

            p(x|s, r, o) =                  N (xi; ris, r2i)[oi=0]N (xi; Ei, Vi)[oi=1],                                                (2)                                            i


where oi {0, 1} is a bernoulli random variable indicating if the measurement at probe xi is the result of the AS model or the outlier model parameterized by p(oi = 1) = i. The parameters of the outlier model, E and V, are not optimized and are set to the mean and variance of the data.

2.2 Variational learning in the GenASAP model

To infer the posterior distribution over the splice isoform abundances while at the same time learning the model parameters we use a variational expectation-maximization algorithm (EM). EM maximizes the log likelihood of the data by iteratively estimating the posterior distribution of the model given the data in the expectation (E) step, and maximizing the log likelihood with respect to the parameters, while keeping the posterior fixed, in the maximization (M) step. Variational EM is used when, as in the case of GenASAP, the exact posterior is intractable. Variational EM minimizes the free energy of the model, defined as the KL-divergence between the joint distribution of the latent and observed variables and the approximation to the posterior under the model parameters [5, 6].

We approximate the true posterior using the Q distribution given by

                                        T          Q({s(t)}, {o(t)}, {r(t)}) =              Q(r(t))Q(o(t)|r(t))          Q(s(t)|o(t), r(t))                                                                                      i     i                                            t=1                            i                                  (3)                                                      T                                         =Z(t)-1            (t)(t)N (s(t); (t)d                                                                                ro , (t)d                                                                                           ro )[s(t)  0],                                                     t=1


where Z is a normalization constant, the superscript d indicates that is constrained to be diagonal, and there are T iid AS events. For computational efficiency, r is selected from a finite set, r {r1, r2, . . . , rC } with uniform probability. The variational free energy is given by

                                                                     Q({s(t)}, {o(t)}, {r(t)})   F(Q, P ) =                      Q({s(t)}, {o(t)}, {r(t)}) log                                              .                                                                      P ({s(t)}, {o(t)}, {r(t)}, {x(t)})                   r    o     s                                                                                                              (4) Variational EM minimizes the free energy by iteratively updating the Q distribution's vari-


ational parameters ((t), (t), (t)d ro , and (t)d ro ) in the E-step, and the model parameters (, , {r1, r2, . . . , rC}, and ) in the M-step. The resulting updates are too long to be shown in the context of this paper and are discussed in detail elsewhere [3]. A few particular points regarding the E-step are worth covering in detail here.

If the prior on s was a full normal distribution, there would be no need for a variational approach, and exact EM is possible. For a truncated normal distribution, however, the mix- ing proportions, Q(r)Q(o|r) cannot be calculated analytically except for the case where s is scalar, necessitating the diagonality constraint. Note that if was allowed to be a full covariance matrix, equation (3) would be the true posterior, and we could find the sufficient statistics of Q(s(t)|o(t), r(t)):

   (t)         ro = (I + T (I - O(t))T -1(I - O(t)))-1T (I - O(t))T -1x(t)r(t)-1                               (5)

(t)-1                             ro      = (I + T (I - O(t))T -1(I - O(t)))                                    (6)


where O is a diagonal matrix with elements Oi,i = oi. Furthermore, it can be easily shown that the optimal settings for d and d approximating a normal distribution with full covariance and mean is

                                              doptimal =                                               (7)

d-1                                            optimal = diag(-1)                                               (8)


In the truncated case, equation (8) is still true. Equation (7) does not hold, though, and doptimal cannot be found analytically. In our experiments, we found that using equation (7) still decreases the free energy every E-step, and it is significantly more efficient than using, for example, a gradient decent method to compute the optimal d.

                              Intuitive Weigh Matrix                                              Optimal Weight Matrix

50                                                               50

40                                                               40

30                                                               30

20                                                               20

10                                                               10

0                                                                    0                                 Inclusion Isoform           Exclusion Isoform                      Inclusion Isoform      Exclusion Isoform                                                      (a)                                                                (b)


Figure 4: (a) An intuitive set of weights. Based on the biological background, one would expect to see the inclusion isoform hybridize to the probes measuring C1, C2, A, C1:A, and A:C2, while the exclusion isoform hybridizes to C1, C2, and C1:C2. (b) The learned set of weights closely agrees with the intuition, and captures cross hybridization between the probes

                                                                                                                           RT-PCR     AS model                               Contribution of         Contribution of                                                    measurement      prediction                                                                                  AS model          Original Data                          exclusion isoform           inclusion isoform                                                   (% exclusion) (% exclusion)

(a)                                                                                                                                14%             27%

(b)                                                                                                           72%             70%

outliers

(c)                                                                                                            8%             22%


Figure 5: Three examples of data cases and their predictions. (a) The data does not follow our notion of single cassette exon AS, but the AS level is predicted accurately by the model.(b) The probe C1:A is marked as outlier, allowing the model to predict the other probes accurately. (c) Two probes are marked as outliers, and the model is still successful in predicting the AS levels.

3 Making biological predictions about alternative splicing

The results presented in this paper were obtained using two stages of learning. In the first step, the weight matrix, , is learned on a subset of the data that is selected for quality. Two selection criteria were used: (a) sequencing data was used to select those cases for which, with high confidence, no other AS event is present (Figure 1) and (b) probe sets were selected for high expression, as determined by a set of negative controls. The second selection criterion is motivated by the common assumption that low intensity measurements are of lesser quality (see Section 1.1). In the second step, is kept fixed, and we introduce the additional constraint that the noise is isotropic ( = I) and learn on the entire data set. The constraint on the noise is introduced to prevent the model from using only a subset of the six probes for making the final set of predictions.

We show a typical learned set of weights in Figure 4. The weights fit well with our intuition of what they should be to capture the presence of the two isoforms. Moreover, the learned weights account for the specific trends in the data. Examples of model prediction based on the microarray data are shown in Figure 5.

Due to the nature of the microarray data, we do not expect all the inferred abundances to be equally good, and we devised a scoring criterion that ranks each AS event based on its fit to the model. Intuitively, given two input vectors that are equivalent up to a scale factor, with inferred MAP estimations that are equal up to the same scale factor, we would like their scores to be identical. The scoring criterion used, therefore is (x k k - rks)2/(xk +

                     Rank     Pearson's correlation      False positive                                         coefficient                 rate                          500               0.94                   0.11                         1000               0.95                   0.08                         2000               0.95                   0.05                         5000               0.79                    0.2                         10000              0.79                   0.25                         15000              0.78                   0.29                         20000              0.75                   0.32                         30000              0.65                   0.42


Table 1: Model performance evaluated at various ranks. Using 180 RT-PCR measurements, we are able to predict the model's performance at various ranks. Two evaluation criteria are used: Pearson's correlation coefficient between the model's predictions and the RT-PCR measurements and false positive rate, where a prediction is considered to be false positive if it is more than 15% away from the RT-PCR measurement.

rks)2, where the MAP estimations for r and s are used. This scoring criterion can be viewed as proportional to the sum of noise to signal ratios, as estimated using the two values given by the observation and the model's best prediction of that observation.

Since it is the relative amount of the isoforms that is of most interest, we need to use the inferred distribution of the isoform abundances to obtain an estimate for the relative levels of AS. It is not immediately clear how this should be done. We do, however, have RT- PCR measurements for 180 AS events to guide us (see figure 6 for details). Using the top 50 ranked RT-PCR measurement, we fit three parameters, {a1, a2, a3}, such that the proportion of excluded isoform present, p, is given by p = a s2 1 + a s 3, where s1 is the 1+a2s2 MAP estimation of the abundance of the inclusion isoform, s2 is the MAP estimation of the abundance of the exclusion isoform, and the RT-PCR measurement are used for target p. The parameters are fitted using gradient descent on a least squared error (LSE) evaluation criterion.

We used two criteria to evaluate the quality of the AS model predictions. Pearson's cor- relation coefficient (PCC) is used to evaluate the overall ability of the model to correctly estimate trends in the data. PCC is invariant to affine transformation and so is independent of the transformation parameters a1 and a3 discussed above, while the parameter a2 was found to effect PCC very little. The PCC stays above 0.75 for the top two thirds ranked pre- dictions. The second evaluation criterion used is the false positive rate, where a prediction is considered to be false positive if it is more than 15% away from the RT-PCR measure- ment. This allows us to say, for example, that if a prediction is within the top 10000, we are 75% confident that it is within 15% of the actual levels of AS.