Abstract A11: Exome sequencing of FFPE material: An evaluation
Authors: Wilzen Annica , Ottesen Heidi , Larsson Anna , Wangberg Bo , Muth Andreas , Nilsson Ola , Abel Frida 
Published: May 2015

Background: The current project aims to explore the possibility to use formalin fixed paraffin embedded (FFPE) samples for exome sequencing, which would open up a large collection of tissue samples for molecular studies. Usage of formalin is known to degrade and modify the DNA both during treatment and storage resulting in problems in downstream molecular analyses. The aim of the present study is to evaluate exome sequencing of FFPE samples by comparing data from FFPE normal tissue to corresponding snap frozen (SF) blood controls with focus on data output such as amount of data removed due to low sequencing quality, amount of data mapped to the genome, GC-content, and duplicate levels.

Methods: In the current study we used the western Swedish biobank of neural tumors archived at the Sahlgrenska Hospital, Gothenburg. We performed exome sequencing of FFPE normal adrenal tissue samples and corresponding SF blood samples from five patients diagnosed with pheochromocytoma/paraganglioma. Two of the patients had known heterozygous germline mutations and were included to determine if these mutations could be found in both sample types. Libraries for SF samples were prepared according to protocol using Agilent Technologies SureSelect Human All Exon 50 Mb library prep kit. FFPE samples were prepared with slight modifications in the protocol, with additional PCR cycles used to increase amplification. 75 bp paired end reads were generated on Illumina HiScan SQ according to manufacturer's protocol (v.3).

Results: Between 92-99% of the raw data were kept after removal of data due to low sequencing quality independent of sample type. For the SF samples 99-100% of the trimmed data mapped to the genome, the number for FFPE samples were slightly lower ranging from 87-98%. The GC content in the exome enriched DNA from FFPE and SF tissue were similar, ranging between 45-51%. Looking at the mapped data we found higher levels of duplicates in the FFPE samples compared to the SF samples, 19-78% vs. 5-15%. For one of the two patients with a known germline mutation the variant was detected at approximately the same frequency in both FFPE and SF sample (39% in FFPE vs. 44% in SF). For the other patient the variant were found at higher frequency in the FFPE sample compared to SF sample (80% vs. 50%).

Conclusions: We saw no difference in amount data removed due to low sequencing quality between the five FFPE and SF samples evaluated in this study. Also, we saw only slightly lower mapping frequencies for the FFPE samples. The GC-content for both FFPE and SF samples were within the range of what's expected for exome sequencing data for libraries prepared with this kit and sequenced on Illumina HiScan SQ platform. Due to the additional PCR cycles used for FFPE samples we saw higher duplicate levels in these samples, suggesting that deeper sequencing is necessary for FFPE samples to get the same amount of unique reads as for the SF samples. A probable explanation to the higher allele frequency of the variant observed in the FFPE sample from one of the patients with a known germline mutation is the presence of contaminant tumor cells in the normal tissue sample, resulting in a shifted allele frequency.

To conclude, SF samples are to prefer for exome sequencing. However, if SF material is not available FFPE tissue is a good alternative to increase the sample cohort when studying rare and complex diseases.

Citation Format: Annica Wilzen, Heidi Ottesen, Anna Larsson, Bo Wangberg, Andreas Muth, Ola Nilsson, Frida Abel. Exome sequencing of FFPE material: An evaluation. [abstract]. In: Proceedings of the AACR Special Conference on Pediatric Cancer at the Crossroads: Translating Discovery into Improved Outcomes; Nov 3-6, 2013; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2013;74(20 Suppl):Abstract nr A11.