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

1. Rashmi Chugh2,
2. Yi-Mi Wu1,
3. Ming Wu1,3,
4. Christine Brennan1,
5. Robert Lonigro1,
6. Fengyun Su1,
7. Rui Wang1,
8. Javed Siddiqui1,
9. Rohit Mehra1,
10. Xuhong Cao1,3,
11. David Lucas4,
12. Arul M. Chinnaiyan1,3,4,5,6,7 and
13. Dan Robinson1,7
14. 1Michigan Center for Translational Pathology, University of Michigan, Ann Arbor, Michigan 48109, USA;
15. 2Department of Internal Medicine, University of Michigan Medical School, Ann Arbor, Michigan 48109, USA;
16. 3Howard Hughes Medical Institute, University of Michigan Medical School, Ann Arbor, Michigan 48109, USA;
17. 4Department of Pathology, University of Michigan Medical School, Ann Arbor, Michigan 48109, USA;
18. 5Comprehensive Cancer Center, University of Michigan Medical School, Ann Arbor, Michigan 48109, USA;
19. 6Department of Urology, University of Michigan Medical School, Ann Arbor, Michigan 48109, USA
20. Corresponding author: arul{at}med.umich.edu

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 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. 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.

Footnotes

• ↵7 Co-senior authors

• [Supplemental material is available for this article.]

• Article published online before print. Article, supplemental material, and publication date are at http://www.genome.org/cgi/doi/10.1101/gr.189621.115.

• Received January 14, 2015.

• Accepted July 15, 2015.

• © 2015 Cieslik et al.; Published by Cold Spring Harbor Laboratory Press

This article is distributed exclusively by Cold Spring Harbor Laboratory Press for the first six months after the full-issue publication date (see http://genome.cshlp.org/site/misc/terms.xhtml). After six months, it is available under a Creative Commons License (Attribution-NonCommercial 4.0 International), as described at http://creativecommons.org/licenses/by-nc/4.0/.