Comparisons of RNA-seq results for cryopreserved (CRYO) and formalin fixed, paraffin-embedded (FFPE) samples.

Abstract

e22211 Background: FFPE is the worldwide clinical standard for preservation of tissue samples. Although CRYO tissues are widely used for research, most gene expression assays in clinical use analyze FFPE by RT-PCR. Use of RT-PCR limits numbers of transcripts that can be studied with material available. Next Generation Sequencing promises to overcome those limitations, but for FFPE, there are concerns related to its accuracy including bias associated with RNA truncated at the 3’ end. Little data is available illustrating the extent of truncation or considering implications for bioinformatics analysis of sequencing results. Methods: Total RNA was extracted from patient consented specimens of tumor and normal bladder tissue split with matching halves processed by CRYO and FFPE. Paired end sequencing libraries 100 bp in length were generated using Illumina TruSeq RNA sample kits with minor modifications for FFPE samples. Sequencing was performed on the Illumina HiSeq 2000, aligned with TopHat using the hg19 reference sequence, and further analyzed using open source bioinformatics tools. Results: Comparing Cryo and FFPE reads for a representative specimen, the proportion of reads that mapped to the reference genome were 69% and 63% for normal, and 66% and 63% for tumor specimens. Mapped reads yielded over 25,000 significantly expressed transcripts, isoforms, and splice variants. The extent of 3’ bias was investigated by viewing data in the Integrated Genome Viewer and using Picard tools. Preliminary estimates of 3’ bias in FFPE showed 2.22 for normal and 1.48 for tumor. After accounting for bias, transcripts in CRYO vs FFPE were significantly correlated for both normal tissue and tumor (R2 = 0.76 and 0.95, respectively). Mutations previously identified in these samples by SureSelect hybrid capture system (Agilent) were also observed in the RNA-seq data. Conclusions: FFPE samples can be successfully analyzed by RNA-Seq, allowing the analysis of standard clinical samples and vast quantities of richly annotated archival specimens. Results from CRYO and FFPE analyses are similar when performed using minor modifications to sequencing library preparation and careful attention to appropriate analysis.