Abstract P1-05-23: Utilities and challenges of RNA-Seq based expression and variant calling in a clinical setting
Authors: Young B , Mark A , Meissner T , Amallraja A , Andrews A , Connolly C , Williams C , Leyland-Jones B 
Published: March 2017

Abstract
Introduction
Variant calling based on DNA samples has been the gold standard of clinical testing since the advent of Sanger sequencing. The use of DNA variants has proved a great value to guide treatment in cancer patients. However, DNA based analysis will not inform about expression status of the gene harboring a particular variant. RNA has long been used to monitor expression. To this point RNA assays and analysis are confined to the research laboratory and rarely used clinically except in specifically defined gene signatures such as PAM50 and OncoType Dx. Beyond expression, RNA has the ability to confirm expression of DNA variants and identify fusion events. We hypothesize that the combination of DNA and RNA based data will allow the determination of variant specific expression status and improve clinical diagnostics. It has been previously shown that RNA sequencing (RNA-Seq) based variant calls are highly accurate and confirm DNA based variant calls. In this study we investigated the utility of RNA-Seq as a diagnostic assay integrated with DNA based sequencing data.
Materials and Methods
Targeted DNA sequencing of 321 genes was performed on 37 patient samples (FFPE), including 22 breast cancer samples by a commercial vendor. RNA-Seq on the same patient samples was performed using 100ng of total RNA. Libraries were run on the Illumina NextSeq 500 with a minimum of 75M paired 75bp reads. To evaluate RNA-seq expression reproducibility, replicates of 6 normal ovarian tissue samples (min. 50M reads) were run in sets of triplicates. STAR was used for alignment (hg19) and gene expression quantification (RefSeq). RNA-Seq based variant calling was performed using the SNPiR pipeline. Based on the results of the commercial assay, DNA based variants were examined for expression of the corresponding genes and ability to confirm variants in the RNA-Seq data.
Results
RNA expression data showed no corresponding gene expression for at least one single nucleotide variant (SNV) in 9/37 patients analyzed (24.3%). In 18/37 patients (48.6%) SNV corresponding expression was in the lowest quartile of expression values. Variant calls could be confirmed by RNA-Seq for 95/455 SNVs, with adequate coverage in 263 of the remaining 360 variant locations (median coverage: 34). Of these, a homozygous reference call was made in 166/263 SNVs. Concordance for RNA-Seq gene level expression data between replicates was &gt; 0.995.
Conclusions
These findings suggest that RNA-Seq based data can provide clinical value when using gene expression values in combination with DNA based variant calls. We found gene level expression to be highly reproducible and will further investigate the use of spike in controls to determine clinically usable expression ranges and lower limit of expression values. To our knowledge, it has not been shown that RNA-Seq based variant calls are reproducible which is the focus of our current research as this will be one requirement for usage in a regulated environment. While our use of RNA Seq is currently limited to gene expression level data, we have demonstrated a clinically relevant benefit to using RNA Seq data as an additive feature to the current standard of DNA variant calling.
Citation Format: Young B, Mark A, Meissner T, Amallraja A, Andrews A, Connolly C, Williams C, Leyland-Jones B. Utilities and challenges of RNA-Seq based expression and variant calling in a clinical setting [abstract]. In: Proceedings of the 2016 San Antonio Breast Cancer Symposium; 2016 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2017;77(4 Suppl):Abstract nr P1-05-23.