Comprehensive alternative splicing recovery needs 200 million reads
Creators
- 1. Universität Hamburg
- 2. University Medicine Greifswald
- 3. Technical University of Munich
- 4. National Institute of Diabetes, Digestive, and Kidney Diseases
- 5. TyrolPath Obrist Brunhuber GMBH
- 6. Kiel University
- 7. Peter L. Reichertz Institute for Medical Informatics of Technische Universität Braunschweig and Hannover Medical School
Description
A key parameter in the experimental design of poly(A) 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, in particular with respect to 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 has only been proposed for human adipose tissue. Alternative splicing, however, differs across tissues and conditions. We analyzed deeply sequenced paired-end RNA-seq samples (between 150 and >500 million reads, read length 50-150 bp) from human buffy coat cells and diverse tissues, including gluteal subcutaneous fat, heart, and hypothalamus. We provide evidence that for genes with moderate and high expression, 200 million reads is the lower bound for comprehensive analysis of alternative splicing. Hence, given the current state of methodology, integrating splicing into the systems biology landscape requires much deeper sequencing than in most published cohorts.
Files
Files
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