The use of exome capture RNA-seq for highly degraded RNA with application to clinical cancer sequencing

Abstract

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, we developed an exomecapture 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. Further, we demonstrate the utility of capture RNA-seq libraries in a patient with a highly malignant solitary fibrous tumor (SFT) enrolled in our clinical sequencing program called MI-ONCOSEQ. Capture transcriptome profiling from FFPE revealed two oncogenic fusions: the pathognomonic NAB2-STAT6 inversion and a therapeutically actionable BRAF fusion, which may drive this specific cancer's aggressive phenotype.

Exome-capture RNA sequencing of decade-old breast cancers and matched decalcified bone metastases

“…This study reinforces and should encourage the use of capture-hybridization approaches to sequence RNA from retrospectively collected, low-yield, highly degraded, and decalcified archival specimens (Supplemental Table 14 and refs. [22][23][24]. Expanding sample sets and modalities for genomewide characterization, especially for rare specimen cohorts that may be impractical to obtain prospectively in large numbers, will accelerate translational discoveries.…”

“…In light of this, new capture-based methods of nucleic acid sequencing on aged FFPE specimens have shown efficacy in identifying DNA variants and even guiding care in academic centers (19)(20)(21). Exome-capture RNA sequencing (ecRNA-seq) is less well characterized in aged tumor samples, although recent studies on FFPE specimens have shown promising expression correlations with flash-frozen tissues (22)(23)(24).…”

The power and promise of RNA‐seq in ecology and evolution

“…those with nontypical RNA profiles, Diaz de Cerio et al 2012), and there is no threshold for deciding a sample is too degraded for whole-transcriptome analysis. Specific library preparation techniques and analytical corrections are available to help optimize the biological signal from degraded but valuable samples whose exclusion would otherwise compromise overall power (Adiconis et al 2013;Romero et al 2014;Cieslik et al 2015). Other well-known sources of technical variance in RNA-seq data are library preparation (e.g.…”

Impact of genomic sequencing on precision medicine for clinical oncology

“…See main text for details on advantages and disadvantages of each strategy. enrichment for research-grade RNA sequencing for the detection and the discovery of gene translocations [16].…”