title: Comparison of Accuracy of Whole-Exome Sequencing with Formalin-Fixed Paraffin-Embedded and Fresh Frozen Tissue Samples.
abstract: Formalin fixing with paraffin embedding (FFPE) has been a standard sample preparation method for decades, and archival FFPE samples are still very useful resources. Nonetheless, the use of FFPE samples in cancer genome analysis using next-generation sequencing, which is a powerful technique for the identification of genomic alterations at the nucleotide level, has been challenging due to poor DNA quality and artificial sequence alterations. In this study, we performed whole-exome sequencing of matched frozen samples and FFPE samples of tissues from 4 cancer patients and compared the next-generation sequencing data obtained from these samples. The major differences between data obtained from the 2 types of sample were the shorter insert size and artificial base alterations in the FFPE samples. A high proportion of short inserts in the FFPE samples resulted in overlapping paired reads, which could lead to overestimation of certain variants; >20% of the inserts in the FFPE samples were double sequenced. A large number of soft clipped reads was found in the sequencing data of the FFPE samples, and about 30% of total bases were soft clipped. The artificial base alterations, C>T and G>A, were observed in FFPE samples only, and the alteration rate ranged from 200 to 1,200 per 1M bases when sequencing errors were removed. Although high-confidence mutation calls in the FFPE samples were compatible to that in the frozen samples, caution should be exercised in terms of the artifacts, especially for low-confidence calls. Despite the clearly observed artifacts, archival FFPE samples can be a good resource for discovery or validation of biomarkers in cancer research based on whole-exome sequencing.
authors.affiliation: Laboratory of Cancer Genomics and Molecular Pathology, Samsung Biomedical Research Institute, Samsung Medical Center, Seoul, Korea.|Institute for Refractory Cancer Research, Samsung Medical Center, Seoul, Korea.|Department of Pharmacy, College of Pharmacy, Seoul National University, Seoul, Korea.
authors.forename: Ensel
authors.initials: E
authors.lastname: Oh
authors.affiliation: Laboratory of Cancer Genomics and Molecular Pathology, Samsung Biomedical Research Institute, Samsung Medical Center, Seoul, Korea.|Institute for Refractory Cancer Research, Samsung Medical Center, Seoul, Korea.|Department of Pathology, Samsung Medical Center, Sungkyunkwan University College of Medicine, Seoul, Korea.|Samsung Advanced Institute for Health Sciences & Technology, Sungkyunkwan University School of Medicine, Seoul, Korea.
authors.forename: Yoon-La
authors.initials: YL
authors.lastname: Choi
authors.affiliation: College of Pharmacy, Kyungpook National University, Daegu, Korea.|Research Institute of Pharmaceutical Sciences, College of Pharmacy, Kyungpook National University, Daegu, Korea.
authors.forename: Mi Jeong
authors.initials: MJ
authors.lastname: Kwon
authors.affiliation: Department of Pharmacy, College of Pharmacy, Seoul National University, Seoul, Korea.
authors.forename: Ryong Nam
authors.initials: RN
authors.lastname: Kim
authors.affiliation: Laboratory of Cancer Genomics and Molecular Pathology, Samsung Biomedical Research Institute, Samsung Medical Center, Seoul, Korea.|Institute for Refractory Cancer Research, Samsung Medical Center, Seoul, Korea.
authors.forename: Yu Jin
authors.initials: YJ
authors.lastname: Kim
authors.affiliation: Laboratory of Cancer Genomics and Molecular Pathology, Samsung Biomedical Research Institute, Samsung Medical Center, Seoul, Korea.|Institute for Refractory Cancer Research, Samsung Medical Center, Seoul, Korea.
authors.forename: Ji-Young
authors.initials: JY
authors.lastname: Song
authors.affiliation: Laboratory of Cancer Genomics and Molecular Pathology, Samsung Biomedical Research Institute, Samsung Medical Center, Seoul, Korea.|Samsung Advanced Institute for Health Sciences & Technology, Sungkyunkwan University School of Medicine, Seoul, Korea.
authors.forename: Kyung Soo
authors.initials: KS
authors.lastname: Jung
authors.affiliation: Department of Pharmacy, College of Pharmacy, Seoul National University, Seoul, Korea.
authors.forename: Young Kee
authors.initials: YK
authors.lastname: Shin
mesh_terms: D015925:Cryopreservation; D004273:DNA, Neoplasm / Q000737:chemistry / Q000235:genetics; D005260:Female; D005557:Formaldehyde / Q000737:chemistry*; D059014:High-Throughput Nucleotide Sequencing / Q000379:methods*; D006801:Humans; D008297:Male; D009369:Neoplasms / Q000737:chemistry / Q000235:genetics* / Q000473:pathology; D016707:Tissue Fixation
chemical_list: D004273:DNA, Neoplasm; D005557:Formaldehyde