Abstract
Abstract Background: To provide a systematic analysis of formalin fixation artifacts on Illumina sequencing libraries and results, we generated two complementary sequencing libraries (target enrichment sequencing and whole exome sequencing) from 11 pairs of matched formalin-fixed paraffin-embedded (FFPE) and fresh-frozen (FF) tumor samples and two pairs of matched FFPE and FF germline samples. We also generated whole genome sequencing data from a single pair of FF/FFPE tumor samples. Results: The results indicate minimal variations in library fragment size, coverage, and PCR duplicates within FF/FFPE paired samples that are less than 1 year old; whereas, a large variation in these parameters were observed in FF/FFPE pairs in samples that are approximately 2 years old. No significant increase in global mismatch rates and C•G>T•A substitutions were observed in FFPE samples from the former group; whereas, a discernible increase in mismatch rates and C•G>T•A substitutions were observed in FFPE samples from the latter group. However, over 99.7% and 99.5% of concordant calls were observed between matched FF and FFPE pairs at reference and non-reference positions within the targeted regions, respectively. Although an increased rate of global mismatches and C•G>T•A substitutions were observed in some FFPE samples, discordant rates were low (<0.26) in all samples because most of the FFPE artifacts were filtered out using GATK workflow. Consistent with this analysis, C•G>T•A substitutions are comparable in non-reference positions in paired FF and FFPE samples. Conclusions: We developed upfront quality assessment and library preparation method that use low input DNA from FFPE samples to perform next-generation sequencing. The results from our studies indicate the suitability of FFPE samples in sequencing studies. Citation Format: Sarah Munchel, Yen Hoang, Yue Zhao, Joseph Cottrell, Brandy Klotzle, Andrew Godwin, Janelle Noel, Brooke Fridley, Peter Beyerlein, Jian-Bing Fan, Marina Bibikova, Jeremy R. Chien. Targeted or whole genome sequencing of formalin-fixed tissue samples. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4281. doi:10.1158/1538-7445.AM2014-4281
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