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. 2009 Apr 16:4:14.
doi: 10.1186/1745-6150-4-14.

Transcript length bias in RNA-seq data confounds systems biology

Alicia Oshlack  1 , Matthew J Wakefield
Affiliations

Transcript length bias in RNA-seq data confounds systems biology

Alicia Oshlack et al. Biol Direct. .

Abstract

Background: Several recent studies have demonstrated the effectiveness of deep sequencing for transcriptome analysis (RNA-seq) in mammals. As RNA-seq becomes more affordable, whole genome transcriptional profiling is likely to become the platform of choice for species with good genomic sequences. As yet, a rigorous analysis methodology has not been developed and we are still in the stages of exploring the features of the data.

Results: We investigated the effect of transcript length bias in RNA-seq data using three different published data sets. For standard analyses using aggregated tag counts for each gene, the ability to call differentially expressed genes between samples is strongly associated with the length of the transcript.

Conclusion: Transcript length bias for calling differentially expressed genes is a general feature of current protocols for RNA-seq technology. This has implications for the ranking of differentially expressed genes, and in particular may introduce bias in gene set testing for pathway analysis and other multi-gene systems biology analyses.

Reviewers: This article was reviewed by Rohan Williams (nominated by Gavin Huttley), Nicole Cloonan (nominated by Mark Ragan) and James Bullard (nominated by Sandrine Dudoit).

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Figures

Figure 1
Figure 1
Differential expression as a function of transcript length. The data is binned according to transcript length and the percentage of transcripts called differentially expressed using a statistical cut-off is plotted (points). A linear regression is also plotted (lines). a – e use all the data from RNA-seq and the microarrays from studies [4-6] respectively. f and g plot 33% of genes with highest expression levels (blue crosses) and 33% of genes with low expression (red triangles) taken from the microarray data for genes which appear on both platforms in [6]. The regression gives a significant trend for the percent of differential expression with transcript length for a, c, d and f and the lowly expressed genes in g. Note that this figure illustrates common data features between disparate experiments and is not a comparison between platforms, methods or experiments.
Figure 2
Figure 2
Mean-variance relationship. Here we show the sample variance across lanes in the liver sample from the Marioni et al[6] data plotted as a function of the mean for each gene (a). Next we have the same data where the tag counts for each gene are divided by the length of the gene (b). The red line fits a linear relationship between the mean and variance for the one third of shortest genes while the blue line is the linear fit to the longest genes. In plot a the fits are very close to the line of equality between mean and variance (black line) which is what would be expected from a Poisson process. In plot b the short genes have higher variance for a given expression level than long genes.
Figure 3
Figure 3
Length of genes found in KEGG pathways significantly over represented with differentially expressed genes. The first box in the plot represents the length of genes found in the four significant categories from both platforms. The second box is the length of genes found in categories significant only in the sequencing data. The third box is the length of all genes in common to both technologies. It can be seen that categories unique to the sequencing data tend to have longer transcripts.
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