Abstract 3938: Massively parallel sequencing of formalin-fixed, paraffin-embedded tissue
Authors: Everson Richard B. , Glatt-Deeley Heather R. , Burian Linda C. , Perpetua Lorrie A. , Hegde Upendra P. , Lambrecht Richard W. , Sanders Mary M. 
Published: August 2014

Abstract
The standard procedure for processing clinical pathology specimens for diagnostic purposes is by formalin-fixation and paraffin-embedding (FFPE). This method is used world-wide for the vast majority of routine histopathology. Nucleic acids in FFPE specimens are fragmented and crosslinked, changes which interfere with their use in many standard array and other molecular analyses. Massively parallel (or deep or next generation) sequencing (MPS) such as that performed by Illumina's Genome Analyzer and HiSeq and Applied Biosystems SOLiD instruments provide sequence data on millions of short fragments of genomic material in a single assay. DNA used for these assays (including cDNA from gene expression preparations) is prepared for analysis by fragmenting it into short segments. While the short size of fragments required, typically less than 500 base pairs, made it likely that MPS could be applied to DNA and RNA extracted from FFPE tissues, there is little data confirming this or directly comparing results from FFPE and cryopreserved (cryo) specimens. For DNA, issues include whether target enrichment procedures can be applied successfully and whether the use of the FFPE samples would increase variability in target coverage. A special concern for RNA analysis is whether variability in the length of transcripts recovered after FFPE processing would bias calling of differentially expressed genes. We used MPS to analyze specimens processed by splitting the sample and processing mirror images of it immediately by cryo and FFPE. Appropriate kits from Qiagen and Roche were used to extract DNA and RNA from sections of cryo and FFPE tissues. Sequencing libraries were produced using procedures from Illumina and Agilent. Sets of six DNA libraries were constructed for each sample: cDNA from RNA (mRNA), genomic DNA (gDNA), and targeted DNA (tDNA) for matching cryo and FFPE specimens. To determine the nature of the length and termination characteristics of the FFPE RNA, RNA assays used standard mRNA-SEQ and were analyzed with Illumina Genome Analyzer (&gt;36 cycle reads). Sequences of FFPE melanoma and bladder cancer from the RNA-SEQ assay generally started from the 3’ side of the exon and extend about 150-250 bp. The frequency of sequences varied widely among exons, and appears to be informative of RNA levels. Analysis of mirrored images of specimens from patients revealed successful analysis of the FFPE tissues, with comparable numbers of million reads for the mRNA-SEQ libraries (1.01 for cryo vs 0.95 FFPE), gDNA (2.38 cryo vs 2.02 FFPE), and tDNA (0.46 million reads for cryo vs 1.39 million for FFPE). Graphical and statistical comparisons will be presented for these and other samples. Our findings confirm the suitability of FFPE specimens for several key MPS analyses, and indicate that the vast stores of archived specimens will be suitable for intensive genomic analyses.
Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3938. doi:10.1158/1538-7445.AM2011-3938