RNA-seq by poly(A) selection is currently the most common protocol for whole transcriptome sequencing as it provides a broad, detailed, and accurate view of the RNA landscape. Unfortunately, the utility of poly(A) libraries is greatly limited when the input RNA is degraded, which is the norm for research tissues and clinical samples, especially when specimens are formalin-fixed.
To facilitate the use of RNA sequencing beyond cell lines and in the clinical setting, researchers at the University of Michigan developed an exome-capture transcriptome protocol with greatly improved performance on degraded RNA. Capture transcriptome libraries enable measuring absolute and differential gene expression, calling genetic variants, and detecting gene fusions. Through validation against gold-standard poly(A) and Ribo-Zero libraries from intact RNA, we show that capture RNA-seq provides accurate and unbiased estimates of RNA abundance, uniform transcript coverage, and broad dynamic range. Unlike poly(A) selection and Ribo-Zero depletion, capture libraries retain these qualities regardless of RNA quality and provide excellent data from clinical specimens including formalin-fixed paraffin-embedded (FFPE) blocks. Systematic improvements across key applications of RNA-seq are shown on a cohort of prostate cancer patients and a set of clinical FFPE samples.
The exome-capture transcriptome protocol. (A) Flow-chart of library preparation protocols. Steps unique to each protocol are highlighted. Enrichment for mRNA occurs at the RNA or cDNA stage, respectively, for poly(A) and capture RNA-seq. (B) Controlled in vitro degradation through cell lysis and warm incubation. VCaP cells were treated with DHT or MDV3100. Intact RNA, RNA integrity number (RIN) 10, was extracted, and libraries were prepared in technical triplicates. In parallel, RNA was degraded by warm incubation for increasing amounts of time. Paired poly(A) and capture libraries were prepared from the same RNA at each degradation level.