Abstract 5686: Long RNA sequencing of human plasma exosomes reveals full coverage of diverse protein coding and long non coding RNA
Authors: Chakrabortty Sudipto K. , Bedford Lisa , Uchiyama Hidefumi , Tadigotla Vasisht , Valentino Michael D. , Grimm Dominik , Chan Dalin , Badola Sunita , Brock Graham , Skog Johan 
Published: July 2017

Abstract
Growing research in oncology implicates exosomes in cell-to-cell communication and various cancer patho-physiologies. Vesicle-mediated signaling is thought to occur via both protein and RNA transport. However, there is a fundamental lack of understanding regarding the long RNA cargo within exosomes. Previous RNASeq studies on exosomes have largely focused on the small RNA fraction, although the clinical biomarker space remains dominated by mRNA. These studies have reported a relatively small proportion and poor transcript coverage of long RNA which has led many to conclude that exosomes only carry short fragments of mRNA and ncRNA.
To address this, we have developed a novel platform specifically designed to perform RNASeq on long RNA from exosomes to efficiently interrogate these as RNA biomarkers. Paired-end sequencing revealed the presence of a wider diversity of annotated mRNA and ncRNAs than previously recognized in exosomes. Employing our whole transcriptome unique molecular indexing strategy, we were able to accurately quantify the number of unique RNA molecules. We detected a total of over 1.4 million unique RNA molecules in our libraries, with 271,308 mapped to protein-coding regions from as little as 2 ml of plasma. We observed that even at a shallow sequencing depth (15 million read pairs), we could detect more than 10,000 genes, composed of 40,000 mRNA and 37,000 ncRNA annotated transcripts. ERCC spike-in analysis indicates that our RNASeq platform is highly sensitive, detecting as few as 8 copies of RNA. Long non-coding RNAs dominated the ncRNA classes and accounted for over 95% of all uniquely detected ncRNAs. We also observed a bimodal distribution of transcript coverage, similar to cellular and tissue derived RNA. In excess of 15,000 transcripts were detected with ≥80% coverage and of these high-coverage transcripts, more than 25% were over 1kb in length, the longest being 17.6kb. Taken together, these findings suggest exosomes harbor more than just small RNA transcripts and short fragments of mRNAs as was previously thought and is a treasure trove of RNA biomarkers.
Current research in the liquid biopsy space has been limited to variant detection in cell-free DNA and circulating tumor cells but transcriptional dysregulation is also an important feature of cancer. Our results shed new light on the biology of exosomes and indicate that, in addition to well-recognized small RNA cargo, plasma exosomes carry an abundance of long RNAs that can be interrogated by our optimized workflow. As exosomes provide a rich and protected source of RNA in biofluids, sequencing both long and small RNA from exosomes has the potential to identify biomarkers without the need for invasive tissue biopsies and allow us to have a more complete picture of how the RNA transcriptome in plasma changes in health and disease.
Citation Format: Sudipto K. Chakrabortty, Lisa Bedford, Hidefumi Uchiyama, Vasisht Tadigotla, Michael D. Valentino, Dominik Grimm, Dalin Chan, Sunita Badola, Graham Brock, Johan Skog. Long RNA sequencing of human plasma exosomes reveals full coverage of diverse protein coding and long non coding RNA [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5686. doi:10.1158/1538-7445.AM2017-5686