Comparison of Accuracy of Whole-Exome Sequencing with Formalin-Fixed Paraffin-Embedded and Fresh Frozen Tissue Samples
نویسندگان
چکیده
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.
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De Paoli-Iseppi, R., Johansson, P., Menzies, A., Dias, K., Pupo, G., Kakavand, H., Wilmott, J., Mann, G., Hayward, N., Dinger, M., Long, G., Scolyer, R. (2016). Comparison of whole-exome sequencing of matched fresh and formalin fixed paraffin embedded melanoma tumours: implications for clinical decision making. Pathology, 48(3), 261-266. ...
متن کاملPublications for Graham
De Paoli-Iseppi, R., Johansson, P., Menzies, A., Dias, K., Pupo, G., Kakavand, H., Wilmott, J., Mann, G., Hayward, N., Dinger, M., Long, G., Scolyer, R. (2016). Comparison of whole-exome sequencing of matched fresh and formalin fixed paraffin embedded melanoma tumours: implications for clinical decision making. Pathology, 48(3), 261-266. ...
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