Abstract 3626: Establishing a robust NGS laboratory workflow and analysis pipeline for FFPE specimen RNAseq to support biopharmaceutical translational research
Authors: Mavrommatis Konstantinos , Intagliata Lauren , McGrath Garth , Civello Daniel , Cronin Maureen 
Published: July 2016

Abstract
Pharmaceutical R&D translational programs rely on analyzing DNA and RNA from patient specimens. High quality histopathology assessment is not possible from fresh and frozen tissues which are also difficult to store long term for follow up studies. FFPE preserves tissue architecture and allows easy archival storage and extracting genomic information from FFPE clinical trial samples is now commonly done, opening a variety of research possibilities. NGS is becoming the preferred approach to FFPE molecular profiling as it generates richer, more complete data than previously possible with microarray technologies.
Our translational research objective was to quantify RNA isoforms for a number of genes requiring that we control for NGS coverage and analysis pipeline biases using embedded controls and comparing multiple calling algorithms. To optimize RNAseq library construction performance using FFPE samples we compared five commercially available rRNA depletion/mRNA selection methods for reducing rRNA abundance: Ovation® (Nugen), Ribo-Zero® (Epicentre), Ribo-Zero GOLD® (Epicentre), RiboGone® (Clonetech) and TruSeq® RNA Access (Illumina). We also evaluated DV200 as a performance metric for predicting NGS library quality for FFPE samples using TruSeq® (Illumina) and SMARTer® (Clonetech) library preparation methods. Finally, we compared RecoverAll® (Life Technologies) and Allprep® (Qiagen) RNA extraction method effects on NGS library quality. By performing the experimental comparisons using two NGS service providers, we were able to assess the impact of laboratory performance on NGS RNAseq results. Ultimately, we were able to define an NGS workflow based on sample QC assessment at multiple points in the process resulting in a highly reliable RNAseq workflow for clinical NGS specimens.
Among the rRNA depletion methods, RiboGone and RNA Access outperformed alternative options. RiboGone virtually eliminated rRNA contamination from FFPE specimens with high reproducibility. RNA Access both eliminated rRNA and provided a high fraction of coding RNA sequences relative to the background of ncRNA allowing experimental designs with lower total read numbers. Depending on experimental requirements, either method is reliable; however, the two methods cannot be used interchangeably without computational normalization. The two library preparation and RNA extraction methods performed similarly in this experiment. DV200 was found to be an good metric for evaluating the quality of FFPE RNA and predicting success in generating RNAseq data.
Our experience across NGS vendors showed that to get optimal results, vendor performance must be managed by using layers of controls with agreed on laboratory performance metrics and explicitly setting NGS experimental parameters in advance of performing the work.
Citation Format: Konstantinos Mavrommatis, Lauren Intagliata, Garth McGrath, Daniel Civello, Maureen Cronin. Establishing a robust NGS laboratory workflow and analysis pipeline for FFPE specimen RNAseq to support biopharmaceutical translational research. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3626.