Abstract 4150: Quantitative sequencing following PCR-driven library preparation with internal standard mixtures has improved analytical performance and lower cost.
Authors: Blomquist Thomas , Crawford Erin L. , Willey James C. 
Published: August 2014

Abstract
Background: Next-generation sequencing (NGS) is amenable to a multitude of clinical applications by virtue of its automated and highly parallelized analysis of nucleic acid templates. However, prior studies have identified non-systematic biases introduced during preparation of NGS libraries as the primary source of technical variation preventing immediate application for measuring nucleic acid abundance in the clinical setting. We reasoned that a PCR-based NGS library preparation protocol that incorporated competitive internal amplification control (IAC) mixtures (i.e. internal standards) would control for the majority of bias introduced during NGS library preparation, enabling clinical laboratories to offer cost effective moderately complex diagnostic panels from quantitative NGS data. Methods: In order to test this approach, we obtained reference material RNA titration pools used in the FDA-sponsored Sequencing Quality Control (SEQC) project that have been characterized for nucleic acid abundance by multiple qPCR, Microarray and NGS platforms. Using Multiplex-PCR with primers and competitive IAC for 150-gene targets we prepared NGS libraries from: 1) gDNA to test general analytical performance, and 2) cDNA from reverse transcribed SEQC project reference material to determine accuracy in detecting fold change. Using gDNA mixed with serially titrated IAC mixtures as input, we observed a linear dynamic range over 106 orders of magnitude, with an average R2 = 0.995 (0.993 – 0.997; 95% CI). There was a high correlation coefficient (R2= 0.96) between measured values for copies of nucleic acid abundance in two separate library preparations (separate reverse transcriptions and Multiplex PCR-based library preparations) from the same reference RNA material (FDA SEQC project Sample A). Because the SEQC project RNA Samples C and D represent a known cross titration between SEQC project RNA Samples A and B, by comparing measured to expected values for expression of each gene in Samples C and D it is possible to determine accuracy of the method. In preliminary studies, the correlation coefficient of expected versus observed for Sample C was R2 = 0.96, with an ROC curve-determined accuracy to detect a 3-fold change of 97% (95 – 99%; 95% CI). Inter-platform and inter-laboratory comparisons are ongoing. Conclusion: The approach described here overcomes key sources of non-systematic bias introduced during NGS library preparation. This should enable reproducible inter-laboratory and inter-platform quantitative NGS results, and a clear path to regulatory approval for clinical diagnostic applications.
Citation Format: Thomas Blomquist, Erin L. Crawford, James C. Willey. Quantitative sequencing following PCR-driven library preparation with internal standard mixtures has improved analytical performance and lower cost. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4150. doi:10.1158/1538-7445.AM2013-4150