RNA sequencing depth guidelines for the study of alternative splicing

Abstract

A key parameter in the experimental design of RNA-seq projects is the choice of sequencing depth. Considering a limited budget, one needs to find a tradeoff between the number of samples and the sensitivity of the analysis, particularly concerning lowly expressed genes. While previous studies have proposed a lower bound for the comprehensive analysis of differential gene expression, for the analysis of alternative splicing, it has only been proposed for human adipose tissue. However, alternative splicing differs across tissues and conditions. We analyzed publicly available and newly generated deep-sequenced paired-end RNA-seq samples (between 150 and >500 million reads, read length 50-150 bp) from human buffy coat cells and diverse sets of tissues, including gluteal subcutaneous fat, heart, and hypothalamus. Our results show that the sequencing depth typically used in published cohorts is not sufficient to comprehensively capture the landscape of alternative splicing. This motivates the use of deeper sequencing or long-read technologies in future studies. Toward this goal, we offer guidelines for choosing a suitable sequencing depth. GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=177 SRC="FIGDIR/small/617406v2_ufig1.gif" ALT="Figure 1"> View larger version (24K): org.highwire.dtl.DTLVardef@1aecd9org.highwire.dtl.DTLVardef@1b3d0fborg.highwire.dtl.DTLVardef@5d15c4org.highwire.dtl.DTLVardef@140035b_HPS_FORMAT_FIGEXP M_FIG C_FIG