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Transcript isoform level expression and differential expression estimation for RNA-seq


In case that the user is only interested to estimate transcript expression and not to proceed to downstream analysis (e.g.: differential expression) we suggest to use the Variational Bayes (VB) version of BitSeq which is significantly faster than MCMC. The VB version of BitSeq approximates the posterior distribution of relative transcript abundance (theta) using a Dirichlet distribution. It has been demonstrated1, 2 that this approximation is almost as accurate as MCMC for estimating the posterior means of transcript expression.

Basic usage

Assume that we have already obtained a data.prob file of read alignments using the parseAlignment command. The following command will estimate the relative transcript expression using our VB algorithm.

$ $BitSeq/estimateVBExpression -o data data.prob

The algorithm produces the file data.m_alphas which contains three columns. The first one corresponds to the estimated relative transcript expression levels (mean theta). The next two columns contain the parameters of the marginal Beta distribution describing the estimated distribution per transcript (alpha, beta). The resulting file will contains M lines, one for each transcript. Note that the joint distribution of all M transcripts is a Dirichlet with the parameters written in the second column (alpha).

RPKM output

In this case we also use the transcript information file produced by the parseAlignment command. Enable the --samples option in order to simulate RPKM values and then summarize the result by calling the getVariance command which provides the estimates of the posterior mean (and variance) of simulated RPKM values per transcript.

$ $BitSeq/estimateVBExpression -o data --outType RPKM -t --samples=1000 data.prob
$ $BitSeq/getVariance -o data.RPKM data.VBrpkm

The first column of the final output file data.RPKM contains the mean RPKM values (one per transcript).


1 Hensman, J., Papastamoulis, P., Glaus, P., Honkela, A., and Rattray, M. (2015). Fast and accurate approximate inference of transcript expression from RNA-seq data. Bioinformatics, 10.1093/bioinformatics/btv483

2 Benchmarking experiments