Abstract 2466: Identifying confidently measured genes in single pediatric cancer patient samples using RNA sequencing
Authors: Beale Holly , Lam Du Linh , Vivian John , Newton Yulia , Shah Avanthi Tayi , Bjork Isabel , Goldstein Ted , Brooks Angela N. , Stuart Josh , Salama Sofie , Sweet-Cordero E. Alejandro , Haussler1 David , Morozova Olena 
Published: July 2017

Abstract
In the UC Santa Cruz Treehouse Childhood Cancer Initiative (treehousegenomics.soe.ucsc.edu), we are exploring the utility of using RNA-Seq analysis of tumor samples from children to identify potential novel therapeutic options for each individual. Within a single RNA-Seq data set, the gene expression measurements are not equally accurate. The identification of activated, druggable pathways requires accurate gene-level expression measurements.
We receive samples from a variety of clinical and research settings, and the quantity and complexity of the available input material and the depth of sequencing differ. These factors inspired us to develop a tool that will allow us to identify accurate measurements in most RNA-Seq samples we receive.
First, we characterized the relationship between depth of sequencing and the accuracy of the gene expression measurement. We analyzed subsets of reads in samples with more than 50 million Uniquely Mapped, Exonic, Non-duplicate (UMEND) reads. UMEND reads typically constitute over 80% of the reads in a high quality experiment with sufficient starting material. We compared gene expression across the subsets of reads to calculate how many UMEND reads are required to produce consistent measurements. We found that, on average, genes expressed at 1-5 TPM in our data require 30 million reads to be accurately measured. For this calculation, we define accuracy as the condition in which 75% of genes are measured to within 25% of the true value.
Secondly, we use these known relationships to identify genes that have been accurately measured in our tumor RNA-Seq samples. For a sample with 15 million UMEND reads, we find that genes expressed above 5 TPM can be accurately measured and are retained. In the first twelve samples analyzed, samples with more than 10 million UMEND reads retained at least 46% of the genes expressed above zero. We exclude as references those samples with fewer than 10 million UMEND reads due to the marked gene loss after thresholding for this group.
Using accurately measured genes allows us to more confidently assess similarity to other samples, identify enriched pathways, and confirm the expression of drug targets and related molecules under consideration. For example, we reconsidered the CDK4 inhibitor Palbociclib in one patient because the expression of RB1, downstream effector required for Palbociclib-mediated tumor cell death, was under our accuracy threshold. Accuracy thresholds can also be used in experiment planning.
Accuracy thresholding allows us to better assess the value of an RNA-Seq data set and, if necessary, identify the subset of genes whose expression can be confidently considered in a clinical setting. Our experience points to the importance of careful quality control in this process.
Citation Format: Holly Beale, Du Linh Lam, John Vivian, Yulia Newton, Avanthi Tayi Shah, Isabel Bjork, Ted Goldstein, Angela N. Brooks, Josh Stuart, Sofie Salama, E. Alejandro Sweet-Cordero, David Haussler1, Olena Morozova. Identifying confidently measured genes in single pediatric cancer patient samples using RNA sequencing [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 2466. doi:10.1158/1538-7445.AM2017-2466