title: 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 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.
authors.affiliation: Michigan Center for Translational Pathology, University of Michigan, Ann Arbor, Michigan 48109, USA;
authors.forename: Marcin
authors.initials: M
authors.lastname: Cieslik
authors.affiliation: Department of Internal Medicine, University of Michigan Medical School, Ann Arbor, Michigan 48109, USA;
authors.forename: Rashmi
authors.initials: R
authors.lastname: Chugh
authors.affiliation: Michigan Center for Translational Pathology, University of Michigan, Ann Arbor, Michigan 48109, USA;
authors.forename: Yi-Mi
authors.initials: YM
authors.lastname: Wu
authors.affiliation: Michigan Center for Translational Pathology, University of Michigan, Ann Arbor, Michigan 48109, USA; Howard Hughes Medical Institute, University of Michigan Medical School, Ann Arbor, Michigan 48109, USA;
authors.forename: Ming
authors.initials: M
authors.lastname: Wu
authors.affiliation: Michigan Center for Translational Pathology, University of Michigan, Ann Arbor, Michigan 48109, USA;
authors.forename: Christine
authors.initials: C
authors.lastname: Brennan
authors.affiliation: Michigan Center for Translational Pathology, University of Michigan, Ann Arbor, Michigan 48109, USA;
authors.forename: Robert
authors.initials: R
authors.lastname: Lonigro
authors.affiliation: Michigan Center for Translational Pathology, University of Michigan, Ann Arbor, Michigan 48109, USA;
authors.forename: Fengyun
authors.initials: F
authors.lastname: Su
authors.affiliation: Michigan Center for Translational Pathology, University of Michigan, Ann Arbor, Michigan 48109, USA;
authors.forename: Rui
authors.initials: R
authors.lastname: Wang
authors.affiliation: Michigan Center for Translational Pathology, University of Michigan, Ann Arbor, Michigan 48109, USA;
authors.forename: Javed
authors.initials: J
authors.lastname: Siddiqui
authors.affiliation: Michigan Center for Translational Pathology, University of Michigan, Ann Arbor, Michigan 48109, USA;
authors.forename: Rohit
authors.initials: R
authors.lastname: Mehra
authors.affiliation: Michigan Center for Translational Pathology, University of Michigan, Ann Arbor, Michigan 48109, USA; Howard Hughes Medical Institute, University of Michigan Medical School, Ann Arbor, Michigan 48109, USA;
authors.forename: Xuhong
authors.initials: X
authors.lastname: Cao
authors.affiliation: Department of Pathology, University of Michigan Medical School, Ann Arbor, Michigan 48109, USA;
authors.forename: David
authors.initials: D
authors.lastname: Lucas
authors.affiliation: Michigan Center for Translational Pathology, University of Michigan, Ann Arbor, Michigan 48109, USA; Howard Hughes Medical Institute, University of Michigan Medical School, Ann Arbor, Michigan 48109, USA; Department of Pathology, University of Michigan Medical School, Ann Arbor, Michigan 48109, USA; Comprehensive Cancer Center, University of Michigan Medical School, Ann Arbor, Michigan 48109, USA; Department of Urology, University of Michigan Medical School, Ann Arbor, Michigan 48109, USA.
authors.forename: Arul M
authors.initials: AM
authors.lastname: Chinnaiyan
authors.affiliation: Michigan Center for Translational Pathology, University of Michigan, Ann Arbor, Michigan 48109, USA;
authors.forename: Dan
authors.initials: D
authors.lastname: Robinson
mesh_terms: D045744:Cell Line, Tumor; D059472:Exome; D020869:Gene Expression Profiling; D015972:Gene Expression Regulation, Neoplastic; D015723:Gene Library; D023281:Genomics / Q000379:methods; D059014:High-Throughput Nucleotide Sequencing; D006801:Humans; D009369:Neoplasms / Q000235:genetics* / Q000473:pathology; D015514:Oncogene Proteins, Fusion / Q000235:genetics; D020871:RNA Stability; D015203:Reproducibility of Results; D017423:Sequence Analysis, RNA; D059467:Transcriptome
chemical_list: D015514:Oncogene Proteins, Fusion