Abstract 1664: Comparison of RNA sequencing data generated from formalin-fixed, paraffin-embedded (FFPE) papillary thyroid carcinoma samples using different library preparation methods

Abstract

Abstract Comparison of RNA sequencing data generated from formalin-fixed, paraffin-embedded (FFPE) papillary thyroid carcinoma samples using different library preparation methods. Julie Dragon, Ramiro Barrantes, Jessica Hoffman, and Scott Tighe. Genomics research requires a significant sample size to provide robust biological signal, leaving researchers clamoring for access to large sample sets. As this research continues to expand into the clinical arena, the demand to sequence RNA from banked tissue samples, such as formalin-fixed paraffin-embedded blocks, is unavoidable. Readily available, these samples represent a wealth of both patients and disease variation. However, recovering DNA and RNA from such samples can be challenging depending on age of the sample block and fixture protocols, which influence nucleic acid degradation and ultimately our ability to synthesize RNA-Seq libraries. To fulfill the need for increased recovery of quality reads, several manufacturers have developed solutions to address these challenges, including FFPE-specific extraction kits, as well as library synthesis and quality control reagents. In this study, we analyzed the quality and outcome of RNA-Seq data generated from RNA from two library synthesis kits applied to FFPE-derived human thyroid tumors with storage times from 4-12 years. The data processing pipeline included adaptor and poor quality base trimming (trimommatic), removal of rRNA reads (sortMeRNA), genomic alignment to hg19 (HiSat2), and read distribution and alignment quality assessment (RNASeqQC) (Adiconis and Levin, 2013). Differential expression was evaluated using DESeq2 (Love et al, 2014). Results indicated that of the reagents tested, it is possible to improve capture of the usable RNA data, and biological signal, with improved control of rRNA depletion and read duplication with newly developed library preparation kits specifically designed for FFPE samples. Moreover, our analysis illustrates some features of the tested synthesis kits that enable one to perform better for FFPE samples with highly varying quality, and quality metrics to consider when evaluating FFPE samples for sequencing. Citation Format: Julie Dragon, Ramiro Barrantes, Jessica Hoffman, Scott Tighe. Comparison of RNA sequencing data generated from formalin-fixed, paraffin-embedded (FFPE) papillary thyroid carcinoma samples using different library preparation methods [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1664.