OUTRIDER – a statistical method for detecting aberrantly expressed genes in RNA sequencing data

RNA sequencing (RNA-seq) is gaining popularity as a complementary assay to genome sequencing for precisely identifying the molecular causes of rare disorders. A powerful approach is to identify aberrant gene expression levels as potential pathogenic events. However, existing methods for detecting aberrant read counts in RNA-seq data either lack assessments of statistical significance, so that establishing cutoffs is arbitrary, or rely on subjective manual corrections for confounders.

Researchers at the Technical University of Munich have developed OUTRIDER (Outlier in RNA-Seq Finder), an algorithm developed to address these issues. The algorithm uses an autoencoder to model read-count expectations according to the gene covariation resulting from technical, environmental, or common genetic variations. Given these expectations, the RNA-seq read counts are assumed to follow a negative binomial distribution with a gene-specific dispersion. Outliers are then identified as read counts that significantly deviate from this distribution. The model is automatically fitted to achieve the best recall of artificially corrupted data. Precision-recall analyses using simulated outlier read counts demonstrated the importance of controlling for covariation and significance-based thresholds. OUTRIDER is open source and includes functions for filtering out genes not expressed in a dataset, for identifying outlier samples with too many aberrantly expressed genes, and for detecting aberrant gene expression on the basis of false-discovery-rate-adjusted p values. Overall, OUTRIDER provides an end-to-end solution for identifying aberrantly expressed genes and is suitable for use by rare-disease diagnostic platforms.

OUTRIDER Overview

(A) Context-dependent outlier detection. The algorithm identifies gene expression outliers whose read counts are significantly aberrant given the covariations typically observed across genes in an RNA-seq dataset. This is illustrated by a read count (left panel, fifth column, second row from the bottom) that is exceptionally high in the context of correlated samples (left six samples) but not in absolute terms for this given gene. To capture commonly seen biological and technical contexts, an autoencoder models covariations in an unsupervised fashion and predicts read-count expectations. Comparing the earlier mentioned read count with these context-dependent expectations reveals that it is exceptionally high (right panel). The lower panels illustrate the distribution of read counts before and after controlling for covariations for the relevant gene. The red dotted lines depict significance cutoffs. (B) Schema showing the differences in the experimental designs for differential expression analyses and outlier detection analyses; relevant analysis packages are mentioned.